β-arrestin effectors and compositions and methods of use thereof

ABSTRACT

This application describes a family of compounds acting as β-arrestin effectors. Such compounds may provide significant therapeutic benefit in the treatment of chronic and acute cardiovascular diseases.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.12/647,810, filed Dec. 28, 2009, which claims priority from U.S.Provisional Patent Application No. 61/141,126, filed on Dec. 29, 2008,each of which is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

This application relates to a family of compounds acting as β-arrestineffectors. Such compounds may provide significant therapeutic benefit inthe treatment of cardiovascular diseases, such as acute heart failure oracute hypertensive crisis.

BACKGROUND

Drugs targeting GPCRs have been developed based on a signaling paradigmin which stimulation of the receptor by an agonist (e.g., angiotensinII) leads to activation of a heterotrimeric “G protein”, which thenleads to second messenger/down-stream signaling (e.g., viadiacylglycerol, inositol-triphosphate, calcium, etc. . . . ) and changesin physiological function (e.g., blood pressure and fluid homeostasis).There is a need for additional drugs that target GPCRs for treatment ofpathology associated with blood pressure and fluid homeostasis.

The foregoing description of related art is not intended in any way asan admission that any of the documents described therein, includingpending United States patent applications, are prior art to the presentinvention. Moreover, the description herein of any disadvantagesassociated with the described products, methods, and/or apparatus, isnot intended to limit the invention. Indeed, aspects of the inventionmay include certain features of the described products, methods, and/orapparatus without suffering from their described disadvantages.

SUMMARY OF THE INVENTION

According to some embodiments, the present invention provides for novelβ-arrestin effectors having the structure:

Xx-Yy-Val-Ww-Zz-Aa-Bb-Cc,

In the structure above, variables Aa, Bb, Cc, Ww, Xx, Yy, and Zz can beselected from the respective groups of chemical or biological moietieslater described in the detailed description. β-arrestin effectorderivatives and mimetics are also provided. Also provided are processesfor preparing the compounds of the invention.

According to some embodiments, the present invention provides for novel3-arrestin effectors having the structure:

Xx-Arg-Val-Ww-Zz-His-Pro-Cc;

or pharmaceutically acceptable salt, solvate, or hydrate thereof. In thestructure above, variables Cc, Ww, Xx, and Zz can be selected from therespective groups of chemical or biological moieties later described inthe detailed description. β-arrestin effector derivatives and mimeticsare also provided. Also provided are processes for preparing thecompounds of the invention.

According to some embodiments, the compounds of the present inventioncomprise the following formula:

Sar-Arg-Val-Ww-Zz-His-Pro-Cc

or pharmaceutically acceptable salt, solvate, or hydrate thereof. In thestructure above, variables Cc, Ww, and Zz can be selected from therespective groups of chemical or biological moieties later described inthe detailed description. β-arrestin effector derivatives and mimeticsare also provided. Also provided are processes for preparing thecompounds of the invention.

According to some embodiments, the compounds of the present inventioncomprise the following formula:

Sar-Arg-Val-Ww-OMTh-His-Pro-Cc,

or pharmaceutically acceptable salt, solvate, or hydrate thereof. In thestructure above, variables Cc, and Ww can be selected from therespective groups of chemical or biological moieties later described inthe detailed description. β-arrestin effector derivatives and mimeticsare also provided. Also provided are processes for preparing thecompounds of the invention.

According to some embodiments the composition of the invention comprisesa peptide or peptide mimetic selected from the group consisting of

a) a peptide or peptide mimetic comprising the sequence of

-   -   Xx-Yy-Val-Ww-Zz-Aa-Bb-Cc,    -   wherein Xx is selected from the group consisting of null,        sarcosine, N-methyl-L-alanine, N-methyl-D-alanine,        N,N-dimethylglycine, L-aspartic acid, D-aspartic acid,        L-glutamic acid, D-glutamic acid, N-methyl-L-aspartic acid,        N-methyl-L-glutamic acid, pyrrolid-1-ylacetic acid, and        morpholin-4-ylacetic acid;    -   Yy is selected from the group consisting of L-arginine, and        L-lysine;    -   Ww is selected from the group consisting of L-isoleucine,        glycine, L-tyrosine, O-methyl-L-tyrosine, L-valine,        L-phenylalanine, 3-hydroxy-L-tyrosine, 2,6-dimethyl-L-tyrosine,        3-fluoro-L-tyrosine, 4-fluorophenyl-L-analine,        2,6-difluoro-L-tyrosine, 3-nitro-L-tyrosine,        3,5-dinitro-L-tyrosine, 3,5-dibromo-L-tyrosine,        3-chloro-L-tyrosine, O-allyl-L-tyrosine, and        3,5-diiodo-L-tyrosine;    -   Zz is selected from the group consisting of L-isoleucine,        L-valine, L-tyrosine, L-glutamic acid, L-phenylalanine,        L-histidine, L-lysine, L-arginine, O-methyl-L-threonine,        D-alanine, and L-norvaline;    -   Aa is selected from the group consisting of L-histidine,        L-histidine-amide, and L-lysine;    -   Bb is selected from the group consisting of L-proline,        L-proline-amide, D-proline, and D-proline-amide; and    -   Cc is selected from the group consisting of null, L-isoleucine,        L-isoleucine-amide, glycine, glycine-amide, L-alanine,        L-alanine-amide, D-alanine, D-phenylalanine, L-norvaline;    -   provided that when Xx is L-Aspartic acid, Cc is not        L-phenylalanine; when Xx is sarcosine, Cc is not L-isoleucine;        when Ww is glycine, Cc is not glycine; when Xx is sarcosine, and        Zz is L-valine, Cc is not L-alanine; and when Xx is sarcosine,        Ww is a L-tyrosine, and Zz is L-isoleucine, Cc is not L-alanine;        b) a peptide or peptide mimetic wherein the members of the        sequence of the peptide or peptide mimetic maintain their        relative positions as they appear in the sequence described in        (a), wherein spacers of between 1 and 3 amino acids or amino        acid analogues are inserted between one or more of the amino        acids or amino acid analogues as described in (a) and wherein        the total length of the peptide or peptide mimetic is between 6        and 25 amino acids and/or amino acid analogues; and        c) a peptide or peptide mimetic that is at least 70% identical        to the peptide or peptide mimetics described in (a).

According to other embodiments, the compositions of the inventioncomprise a peptide or peptide mimetic selected from the group consistingof

a) a peptide or peptide mimetic comprising the sequence of

Xx-Arg-Val-Ww-Zz-His-Pro-Cc,

-   -   wherein Xx is selected from the group consisting of sarcosine,        N-methyl-L-alanine, N-methyl-D-alanine, N,N-dimethylglycine,        L-aspartic acid, D-aspartic acid, L-glutamic acid, D-glutamic        acid, N-methyl-L-aspartic acid, N-methyl-L-glutamic acid,        pyrrolid-1-ylacetic acid, and morpholin-4-ylacetic acid;    -   Ww is selected from the group consisting of L-isoleucine,        glycine, L-tyrosine, O-methyl-L-tyrosine, L-valine,        L-phenylalanine, 3-hydroxy-L-tyrosine, 2,6-dimethyl-L-tyrosine,        3-fluoro-L-tyrosine, 4-fluorophenyl-L-analine,        2,6-difluoro-L-tyrosine, 3-nitro-L-tyrosine,        3,5-dinitro-L-tyrosine, 3,5-dibromo-L-tyrosine,        3-chloro-L-tyrosine, O-allyl-L-tyrosine, and        3,5-diiodo-L-tyrosine;    -   Zz is selected from the group consisting of L-isoleucine,        L-valine, L-tyrosine, L-glutamic acid, L-phenylalanine,        L-histidine, L-lysine, L-arginine, O-methyl-L-threonine,        D-alanine, and L-norvaline; and    -   Cc is selected from the group consisting of L-isoleucine,        L-isoleucine-amide, glycine, glycine-amide, L-alanine,        L-alanine-amide, D-alanine, D-phenylalanine, and L-norvaline;    -   provided that when Xx is L-Aspartic acid, Cc is not        L-phenylalanine; when Xx is sarcosine, Cc is not L-isoleucine;        when Ww is glycine, Cc is not glycine; when Xx is sarcosine, and        Zz is L-valine, Cc is not L-alanine; and when Xx is sarcosine,        Ww is a L-tyrosine, and Zz is L-isoleucine, Cc is not L-alanine;        b) a peptide or peptide mimetic wherein the members of the        sequence of the peptide or peptide mimetic maintain their        relative positions as they appear in the sequence described in        (a), wherein spacers of between 1 and 3 amino acids or amino        acid analogues are inserted between one or more of the amino        acids or amino acid analogues as described in (a) and wherein        the total length of the peptide or peptide mimetic is between 8        and 25 amino acids and/or amino acid analogues; and        c) a peptide or peptide mimetic that is at least 70% identical        to the peptide or peptide mimetics described in (a).

According to other embodiments, the compositions of the inventioncomprise a peptide or peptide mimetic selected from the group consistingof

a) a peptide or peptide mimetic comprising the sequence of

Sar-Arg-Val-Ww-Zz-His-Pro-Cc,

-   -   wherein Ww is selected from the group consisting of L-tyrosine,        3-hydroxy-L-tyrosine, 3-fluoro-L-tyrosine,        2,6-difluoro-L-tyrosine, 3-nitro-L-tyrosine,        3,5-dinitro-L-tyrosine, and 3-chloro-L-tyrosine;    -   Zz is selected from the group consisting of L-isoleucine,        L-lysine, and O-Methyl-L-threonine; and    -   Cc is selected from the group consisting of D-alanine, and        L-alanine    -   provided that when Xx is sarcosine, Ww is a L-tyrosine, and Zz        is L-isoleucine, Cc is not L-alanine;        b) a peptide or peptide mimetic wherein the members of the        sequence of the peptide or peptide mimetic maintain their        relative positions as they appear in the sequence described in        (a), wherein spacers of between 1 and 3 amino acids or amino        acid analogues are inserted between one or more of the amino        acids or amino acid analogues as described in (a) and wherein        the total length of the peptide or peptide mimetic is between 8        and 25 amino acids and/or amino acid analogues; and        c) a peptide or peptide mimetic that is at least 70% identical        to the peptide or peptide mimetics described in (a).

In more specific embodiments, Ww is selected from the group consistingof L-tyrosine and 3-hydroxy-L-tyrosine; and Zz is selected from thegroup consisting of L-isoleucine and L-lysine. Optionally, the peptideor peptide mimetic comprises the sequence of SEQ ID NO:23, 27 or 67.

According to other embodiments, the compositions of the inventioncomprise a peptide or peptide mimetic selected from the group consistingof

a) a peptide or peptide mimetic comprising the sequence of

Sar-Arg-Val-Ww-OMTh-His-Pro-Cc,

-   -   wherein Ww is selected from the group consisting of L-tyrosine,        3-hydroxy-L-tyrosine; 3-fluoro-L-tyrosine, and        3-chloro-L-tyrosine; and    -   Cc is selected from the group consisting of D-alanine and        L-alanine;        b) a peptide or peptide mimetic wherein the members of the        sequence of the peptide or peptide mimetic maintain their        relative positions as they appear in the sequence described in        (a), wherein spacers of between 1 and 3 amino acids or amino        acid analogues are inserted between one or more of the amino        acids or amino acid analogues as described in (a) and wherein        the total length of the peptide or peptide mimetic is between 8        and 25 amino acids and/or amino acid analogues; and        c) a peptide or peptide mimetic that is at least 70% identical        to the peptide or peptide mimetics described in (a).

In more specific embodiments, Ww is selected from the group consistingof 3-hydroxy-L-tyrosine; 3-fluoro-L-tyrosine, and 3-chloro-L-tyrosine;and Cc is L-alanine. Optionally, the peptide or peptide mimeticcomprises the sequence of SEQ ID NO:77, 78 or 79.

According to other embodiments, the compositions of the inventioncomprise a peptide or peptide mimetic selected from the group consistingof

a) a peptide or peptide mimetic comprising the sequence of

Sar-Arg-Val-Ww-Tyr-His-Pro-NH₂,

-   -   wherein Ww is selected from the group consisting of L-tyrosine,        3-hydroxy-L-tyrosine, 3-fluoro-L-tyrosine,        2,6-difluoro-L-tyrosine, 3-nitro-L-tyrosine,        3,5-dinitro-L-tyrosine, and 3-chloro-L-tyrosine;        b) a peptide or peptide mimetic wherein the members of the        sequence of the peptide or peptide mimetic maintain their        relative positions as they appear in the sequence described in        (a), wherein spacers of between 1 and 3 amino acids or amino        acid analogues are inserted between one or more of the amino        acids or amino acid analogues as described in (a) and wherein        the total length of the peptide or peptide mimetic is between 7        and 25 amino acids and/or amino acid analogues; and        c) a peptide or peptide mimetic that is at least 70% identical        to the peptide or peptide mimetics described in (a).

Optionally, the peptide or peptide mimetic comprises the sequence of SEQID NO:26.

According to other embodiments, the compositions of the inventioncomprise a peptide or peptide mimetic selected from the group consistingof

a) a peptide or peptide mimetic comprising the sequence of

NMAla-Arg-Val-Ww-Zz-His-Pro-Cc,

-   -   wherein Ww is selected from the group consisting of L-tyrosine,        3-hydroxy-L-tyrosine, 3-fluoro-L-tyrosine,        2,6-difluoro-L-tyrosine, 3-nitro-L-tyrosine,        3,5-dinitro-L-tyrosine, and 3-chloro-L-tyrosine;    -   Zz is selected from the group consisting of L-isoleucine,        L-lysine, and O-Methyl-L-threonine;    -   and Cc is selected from the group consisting of D-alanine, and        L-alanine;        b) a peptide or peptide mimetic wherein the members of the        sequence of the peptide or peptide mimetic maintain their        relative positions as they appear in the sequence described in        (a), wherein spacers of between 1 and 3 amino acids or amino        acid analogues are inserted between one or more of the amino        acids or amino acid analogues as described in (a) and wherein        the total length of the peptide or peptide mimetic is between 8        and 25 amino acids and/or amino acid analogues; and        c) a peptide or peptide mimetic that is at least 70% identical        to the peptide or peptide mimetics described in (a).

In more specific embodiments, Ww is L-tyrosine, Zz is L-isoleucine,and/or Cc is L-alanine. Optionally, the peptide or peptide mimeticcomprises the sequence of SEQ ID NO:34.

Optionally, any of the above described compositions of the invention arecyclic, dimerized and/or trimerized.

According to other embodiments, the compositions of the inventioncomprise an angiotensin 2 type 1 receptor (AT1R) β-arrestin biasedligand. In specific embodiments, the ligand has a lower half maximaleffective concentration for recruiting β-arrestin-2 to an angiotensin 2type 1 receptor than for IP1 accumulation. In more specific embodiments,the half maximal effective concentration for recruiting β-arrestin-2 toan angiotensin 2 type 1 receptor of the ligand is ten times lower thanthat for IP1 accumulation of the ligand. In more specific embodiments,the ligand is a composition comprising a peptide or peptide mimeticselected from the group consisting of

a) a peptide or peptide mimetic comprising the sequence of

-   -   Xx-Yy-Val-Ww-Zz-Aa-Bb-Cc,    -   wherein Xx is selected from the group consisting of null,        sarcosine, N-methyl-L-alanine, N-methyl-D-alanine,        N,N-dimethylglycine, L-aspartic acid, D-aspartic acid,        L-glutamic acid, D-glutamic acid, N-methyl-L-aspartic acid,        N-methyl-L-glutamic acid, pyrrolid-1-ylacetic acid, and        morpholin-4-ylacetic acid;    -   Yy is selected from the group consisting of L-arginine, and        L-lysine;    -   Ww is selected from the group consisting of L-isoleucine,        glycine, L-tyrosine, O-methyl-L-tyrosine, L-valine,        L-phenylalanine, 3-hydroxy-L-tyrosine, 2,6-dimethyl-L-tyrosine,        3-fluoro-L-tyrosine, 4-fluorophenyl-L-analine,        2,6-difluoro-L-tyrosine, 3-nitro-L-tyrosine,        3,5-dinitro-L-tyrosine, 3,5-dibromo-L-tyrosine,        3-chloro-L-tyrosine, O-allyl-L-tyrosine, and        3,5-diiodo-L-tyrosine;    -   Zz is selected from the group consisting of L-isoleucine,        L-valine, L-tyrosine, L-glutamic acid, L-phenylalanine,        L-histidine, L-lysine, L-arginine, O-methyl-L-threonine,        D-alanine, and L-norvaline;    -   Aa is selected from the group consisting of L-histidine,        L-histidine-amide, and L-lysine;    -   Bb is selected from the group consisting of L-proline,        L-proline-amide, D-proline, and D-proline-amide; and    -   Cc is selected from the group consisting of null, L-isoleucine,        L-isoleucine-amide, glycine, glycine-amide, L-alanine,        L-alanine-amide, D-alanine, D-phenylalanine, L-norvaline;    -   provided that when Xx is L-Aspartic acid, Cc is not        L-phenylalanine; when Xx is sarcosine, Cc is not L-isoleucine;        when Ww is glycine, Cc is not glycine; when Xx is sarcosine, and        Zz is L-valine, Cc is not L-alanine; and when Xx is sarcosine,        Ww is a L-tyrosine, and Zz is L-isoleucine, Cc is not L-alanine;        b) a peptide or peptide mimetic wherein the members of the        sequence of the peptide or peptide mimetic maintain their        relative positions as they appear in the sequence described in        (a), wherein spacers of between 1 and 3 amino acids or amino        acid analogues are inserted between one or more of the amino        acids or amino acid analogues as described in (a) and wherein        the total length of the peptide or peptide mimetic is between 6        and 25 amino acids and/or amino acid analogues; and        c) a peptide or peptide mimetic that is at least 70% identical        to the peptide or peptide mimetics described in (a).

In more specific embodiments, the ligand is a composition comprising apeptide or peptide mimetic selected from the group consisting of

a) a peptide or peptide mimetic comprising the sequence of

Xx-Arg-Val-Ww-Zz-His-Pro-Cc,

-   -   wherein Xx is selected from the group consisting of sarcosine,        N-methyl-L-alanine, N-methyl-D-alanine, N,N-dimethylglycine,        L-aspartic acid, D-aspartic acid, L-glutamic acid, D-glutamic        acid, N-methyl-L-aspartic acid, N-methyl-L-glutamic acid,        pyrrolid-1-ylacetic acid, and morpholin-4-ylacetic acid;    -   Ww is selected from the group consisting of L-isoleucine,        glycine, L-tyrosine, O-methyl-L-tyrosine, L-valine,        L-phenylalanine, 3-hydroxy-L-tyrosine, 2,6-dimethyl-L-tyrosine,        3-fluoro-L-tyrosine, 4-fluorophenyl-L-analine,        2,6-difluoro-L-tyrosine, 3-nitro-L-tyrosine,        3,5-dinitro-L-tyrosine, 3,5-dibromo-L-tyrosine,        3-chloro-L-tyrosine, O-allyl-L-tyrosine, and        3,5-diiodo-L-tyrosine;    -   Zz is selected from the group consisting of L-isoleucine,        L-valine, L-tyrosine, L-glutamic acid, L-phenylalanine,        L-histidine, L-lysine, L-arginine, O-methyl-L-threonine,        D-alanine, and L-norvaline; and    -   Cc is selected from the group consisting of L-isoleucine,        L-isoleucine-amide, glycine, glycine-amide, L-alanine,        L-alanine-amide, D-alanine, D-phenylalanine, and L-norvaline;    -   provided that when Xx is L-Aspartic acid, Cc is not        L-phenylalanine; when Xx is sarcosine, Cc is not L-isoleucine;        when Ww is glycine, Cc is not glycine; when Xx is sarcosine, and        Zz is L-valine, Cc is not L-alanine; and when Xx is sarcosine,        Ww is a L-tyrosine, and Zz is L-isoleucine, Cc is not L-alanine;        b) a peptide or peptide mimetic wherein the members of the        sequence of the peptide or peptide mimetic maintain their        relative positions as they appear in the sequence described in        (a), wherein spacers of between 1 and 3 amino acids or amino        acid analogues are inserted between one or more of the amino        acids or amino acid analogues as described in (a) and wherein        the total length of the peptide or peptide mimetic is between 8        and 25 amino acids and/or amino acid analogues; and        c) a peptide or peptide mimetic that is at least 70% identical        to the peptide or peptide mimetics described in (a).

According to other embodiments, the invention provides a pharmaceuticalcomposition comprising one or more of the above described peptides orpeptide mimetics and a pharmaceutically acceptable carrier. In specificembodiments, the pharmaceutically acceptable carrier is pure sterilewater, phosphate buffered saline or an aqueous glucose, solution.

According to other embodiments, the invention provides a method oftreating cardiovascular disorders comprising: administering to a subjectin need thereof a therapeutically effective amount of one or morecompositions described above. In specific embodiments, thecardiovascular disorder is selected from the groups consisting ofchronic hypertension, hypertensive crisis, acute congestive heartfailure, angina, acute myocardial infarction, left ventricular failure,cerebrovascular insufficiency, intracranial haemorrhage, heart failure,acute decompensated heart failure, essential hypertension,post-operative hypertension, hypertensive heart disease, hypertensiverenal disease, renovascular hypertension, malignant hypertension,post-renal transplant patient stabilization, dilated cardiomyopathy,myocarditis, post-cardiac transplant patient stabilization, disordersassociated with post-stent management, neurogenic hypertension,pre-eclampsia, abdominal aortic aneurysm, and any cardiovasculardisorder with a hemodynamic component. In more specific embodiments, thecardiovascular disorder is an acute cardiovascular disorder. In otherspecific embodiments, the acute cardiovascular disorder is acutehypertensive crisis, toxemia of pregnancy, acute myocardial infarction,acute congestive heart failure, acute ischaemic heart disease, pulmonaryhypertension, post-operative hypertension, migraine, retinopathy andpost-operative cardiac/valve surgery.

According to other embodiments, the invention also provides a method oftreating viral infectious disease linked to AT1R comprising:administering to a subject in need thereof a therapeutically effectiveamount of one or more compositions described above. In specificembodiments, the one or more compositions are administered byintravenous injection.

According to other embodiments, the invention also provides a method oftreating cardiovascular disorders comprising: administering to a subjectin need thereof a therapeutically effective amount of one or more apeptides or peptide mimetics comprising the sequence of

-   -   Sar-Arg-Val-Ww-Zz-His-Pro-Cc,    -   wherein Ww is selected from the group consisting of        L-isoleucine, glycine, and L-tyrosine;    -   Zz is selected from the group consisting of L-valine,        L-isoleucine and L-glutamate; and    -   Cc is selected from the group consisting of L-isoleucine,        glycine, and L-alanine.

In specific embodiments, the one or more peptides or peptide mimeticsare selected from the group consisting of SEQ ID NOs: 3, 5, 7, 9, 11,14, and 16. in more specific embodiments, the cardiovascular disorder isselected from the groups consisting of chronic hypertension,hypertensive crisis, acute congestive heart failure, angina, acutemyocardial infarction, left ventricular failure, cerebrovascularinsufficiency, intracranial haemorrhage, heart failure, acutedecompensated heart failure, essential hypertension, post-operativehypertension, hypertensive heart disease, hypertensive renal disease,renovascular hypertension, malignant hypertension, post-renal transplantpatient stabilization, dilated cardiomyopathy, myocarditis, post-cardiactransplant patient stabilization, disorders associated with post-stentmanagement, neurogenic hypertension, pre-eclampsia, abdominal aorticaneurysm, and any cardiovascular disorder with a hemodynamic component.In other specific embodiments, the cardiovascular disorder is an acutecardiovascular disorder. In other specific embodiments, the acutecardiovascular disorder is acute hypertensive crisis, toxemia ofpregnancy, acute myocardial infarction, acute congestive heart failure,acute ischaemic heart disease, pulmonary hypertension, post-operativehypertension, migraine, retinopathy and post-operative cardiac/valvesurgery.

According to other embodiments, the invention also provides a method oftreating viral infectious disease linked to AT1R comprising:administering to a subject in need thereof a therapeutically effectiveamount of one or more a peptides or peptide mimetics comprising thesequence of

-   -   Sar-Arg-Val-Ww-Zz-His-Pro-Cc,    -   wherein Ww is selected from the group consisting of        L-isoleucine, glycine, and L-tyrosine;    -   Zz is selected from the group consisting of L-valine,        L-isoleucine and L-glutamate; and    -   Cc is selected from the group consisting of L-isoleucine,        glycine, and L-alanine.

In specific embodiments, the one or more peptides or peptide mimeticsare selected from the group consisting of SEQ ID NOs: 3, 5, 7, 9, 11,14, and 16. According to other embodiments, the invention also providesa method of agonizing β-arrestin comprising: administering to a subjectin need thereof an effective amount of one or more compositionsdescribed above.

According to some embodiments, the present invention extends to apharmaceutical composition comprising a compound of the invention and apharmaceutically acceptable carrier. Naturally, the compounds of thepresent invention can be employed in any form, such as a solid orsolution (e.g., aqueous solution) as is described further below. Thecompound, for example, can be obtained and employed in a lyophilizedform alone or with suitable additives.

Also provided are methods for treating cardiovascular disorders. Such amethod would comprise administering a therapeutically effective amountof one or more compounds of the present invention to a subject in needthereof.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Before the present proteins, nucleotide sequences, peptides, etc., andmethods are described, it is understood that this invention is notlimited to the particular methodology, protocols, cell lines, vectors,and reagents described, as these may vary. It also is to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to limit the scope ofthe present invention which will be limited only by the appended claims.

This application describes a family of compounds, β-arrestin effectors,with a unique profile. The compounds of the present invention act asagonists of β-arrestin/GRK-mediated signal transduction via the AT1angiotensin receptor. Thus, these compounds stimulate signaling pathwaysthat provide significant therapeutic benefit in the treatment ofcardiovascular diseases such as acute heart failure or acutehypertensive crisis.

According to some embodiments, the compounds of the present inventioncomprise the following formula:

Xx-Yy-Val-Ww-Zz-Aa-Bb-Cc,

wherein Xx is null, sarcosine, N-methyl-L-alanine, N-methyl-D-alanine,N,N-dimethylglycine, L-aspartic acid, D-aspartic acid, L-glutamic acid,D-glutamic acid, N-methyl-L-aspartic acid, N-methyl-L-glutamic acid,pyrrolid-1-ylacetic acid, or morpholin-4-ylacetic acid; Yy isL-arginine, or L-lysine; Ww is L-isoleucine, glycine, L-tyrosine,O-methyl-L-tyrosine, L-valine, L-phenylalanine, 3-hydroxy-L-tyrosine,2,6-dimethyl-L-tyrosine, 3-fluoro-L-tyrosine, 4-fluorophenyl-L-analine,2,6-difluoro-L-tyrosine, 3-nitro-L-tyrosine, 3,5-dinitro-L-tyrosine,3,5-dibromo-L-tyrosine, 3-chloro-L-tyrosine, O-allyl-L-tyrosine, or3,5-diiodo-L-tyrosine; Zz is L-isoleucine, L-valine, L-tyrosine,L-glutamic acid, L-phenylalanine, L-histidine, L-lysine, L-arginine,O-methyl-L-threonine, D-alanine, or L-norvaline; Aa is L-histidine,L-histidine-amide, or L-lysine; Bb is L-proline, L-proline-amide,D-proline, D-proline-amide and Cc is null, L-isoleucine,L-isoleucine-amide, glycine, glycine-amide, L-alanine, L-alanine-amide,D-alanine, D-phenylalanine, L-norvaline;provided that when Xx is L-aspartic acid, Cc is not L-phenylalanine;provided that when Xx is sarcosine, Cc is not L-isoleucine;provided that when Ww is glycine, Cc is not glycine;provided that when Xx is sarcosine, and Zz is L-valine, Cc is notL-alanine;provided that when Xx is sarcosine, Ww is a L-tyrosine, and Zz isL-isoleucine, Cc is not L-alanine;

According to some embodiments, the compounds of the present inventioncomprise the following formula:

Xx-Arg-Val-Ww-Zz-His-Pro-Cc,

wherein Xx is sarcosine, N-methyl-L-alanine, N-methyl-D-alanine,N,N-dimethylglycine, L-aspartic acid, D-aspartic acid, L-glutamic acid,D-glutamic acid, N-methyl-L-aspartic acid, N-methyl-L-glutamic acid,pyrrolid-1-ylacetic acid, or morpholin-4-ylacetic acid; Ww isL-isoleucine, glycine, L-tyrosine, O-methyl-L-tyrosine, L-valine,L-phenylalanine, 3-hydroxy-L-tyrosine, 2,6-dimethyl-L-tyrosine,3-fluoro-L-tyrosine, 4-fluorophenyl-L-analine, 2,6-difluoro-L-tyrosine,3-nitro-L-tyrosine, 3,5-dinitro-L-tyrosine, 3,5-dibromo-L-tyrosine,3-chloro-L-tyrosine, O-allyl-L-tyrosine, or 3,5-diiodo-L-tyrosine; Zz isL-isoleucine, L-valine, L-tyrosine, L-glutamic acid, L-phenylalanine,L-histidine, L-lysine, L-arginine, O-methyl-L-threonine, D-alanine, orL-norvaline; and Cc is L-isoleucine, L-isoleucine-amide, glycine,glycine-amide, L-alanine, L-alanine-amide, D-alanine, D-phenylalanine,L-norvaline.provided that when Xx is L-aspartic acid, Cc is not L-phenylalanine;provided that when Xx is sarcosine, Cc is not L-isoleucine;provided that when Ww is glycine, Cc is not glycine;provided that when Xx is sarcosine, and Zz is L-valine, Cc is notL-alanine;provided that when Xx is sarcosine, Ww is a L-tyrosine, and Zz isL-isoleucine, Cc is not L-alanine;

According to some embodiments, the compounds of the present inventioncomprise the following formula:

Sar-Arg-Val-Ww-Zz-His-Pro-Cc,

wherein Ww is L-tyrosine, 3-hydroxy-L-tyrosine, 3-fluoro-L-tyrosine,2,6-difluoro-L-tyrosine, 3-nitro-L-tyrosine, 3,5-dinitro-L-tyrosine,3-chloro-L-tyrosine; Zz is L-valine or L-isoleucine; and Cc isD-alanine.

According to some embodiments, the compounds of the present inventioncomprise the following formula:

Sar-Arg-Val-Ww-OMTh-His-Pro-Cc,

wherein Ww is L-tyrosine, 3-fluoro-L-tyrosine, 3-chloro-L-tyrosine; andCc is D-alanine or L-alanine.

The definition of some of the abbreviations used in the presentinvention is given below:

Chemical name of amino acid Structure of amino acid or Abbreviation orits analog its analog Ala L-Alanine

D-Ala D-Alanine

NMAla N-Methyl-L- alanine

NM-D-Ala N-Methyl-D- alanine

Me2G N,N- dimethylglycine

Asp L-Aspartic acid

D-Asp D-Aspartic acid

Glu L-Glutamic acid

D-Glu D-Glutamic acid

N—Me-Asp N-Methyl-L- aspartic acid

N—Me-Glu N-Methyl-L- glutamic acid

Sar Sarcosine

MMM Pyrrolid-1- ylacetic acid

NNN Morpholin-4- ylacetic acid

Arg L-Arginine

Lys L-Lysine

ILe L-Isoleucine

Gly Glycine

Tyr L-Tyrosine

OMTyr O-Methyl-L- tyrosine

Val L-Valine

Phe L- Phenylalanine

D-Phe D- Phenylalanine

AAA 3-Hydroxy-L- tyrosine

BBB 2,6-Dimethyl- L-tyrosine

CCC 3-Fluoro-L- tyrosine

DDD 4- Fluorophenyl- L-alanine

EEE 2,6-Difluoro-L- tyrosine

FFF 3-Nitro-L- tyrosine

GGG 3,5-Dinitro-L- tyrosine

HHH 3,5-Dibromo-L- tyrosine

III 3-Chloro-L- tyrosine

JJJ O-Allyl-L- tyrosine

OOO 3,5-Diiodo-L- tyrosine

His L-Histidine

OMTh O-Methyl-L- threonine

NVA L-Norvaline

Pro L-Proline

D-Pro D-Proline

L-Histidine- amide

L-Proline amide

D-Proline amide

L-Isoleucine amide

Glycine amide

L-Alanine amide

Cyclic forms, cyclic truncated forms, cyclic truncated dimerized forms,and cyclic truncated trimerized forms of the compounds of the aboveformulas may be prepared using any known method. Truncated is defined asanalogs with amino acids removed from the X1 and/or X2 residues asdepicted in Table 1. According to some embodiments, cyclic forms of thecompounds of the above formulas may be prepared by bridging free aminoand free carboxyl groups. According to some embodiments, formation ofthe cyclic compounds may be conducted conventionally by treatment with adehydrating agent by means known in the art, with suitable protection ifneeded. According to some embodiments, the open chain (linear form) tocyclic form reaction may involve a trans to cis isomerization of theproline. According to some embodiments, the open chain (linear form) tocyclic form reaction may involve intramolecular-cyclization.

Examples of the compounds of the present invention include, but are notlimited to, the compounds listed in Table 1 below.

TABLE 1  SEQ ID Residue (X) NO: X₁ X₂ X₃ X₄ X₅ X₆ X₇ X₈ AngII Asp ArgVal Tyr Ile His Pro Phe OH 2 Sar Arg Val Ile Ile His Pro Ile NH2 3 SarArg Val Ile Val His Pro Ile OH 5 Sar Arg Val Gly Val His Pro Gly OH 6Sar Arg Val Gly Val His Pro Gly NH2 7 Sar Arg Val Tyr Val His Pro Ala OH8 Sar Arg Val Tyr Val His Pro Ala NH2 9 Sar Arg Val Tyr Ile His Pro AlaOH 10 Sar Arg Val Tyr Ile His Pro Ala NH2 11 Sar Arg Val Tyr Val His ProIle OH 12 Sar Arg Val Tyr Val His Pro Ile NH2 13 Arg Val Tyr Ile His ProNH2 14 Sar Arg Val Tyr Tyr His Pro Ile OH 15 Sar Arg Val Tyr Tyr His ProIle NH2 16 Sar Arg Val Tyr Glu His Pro Ile OH 19 Sar Arg Val Tyr Tyr HisPro Ala OH 20 Sar Arg Val Tyr Glu His Pro Ala OH 21 Sar Arg Val Tyr PheHis Pro Ala OH 22 Sar Arg Val Tyr His His Pro Ala OH 23 Sar Arg Val TyrLys His Pro Ala OH 24 Sar Arg Val Tyr Arg His Pro Ala OH 25 Asp Arg ValTyr Ile His Pro Ala OH 26 Sar Arg Val Tyr Tyr His Pro NH2 27 Sar Arg ValTyr Ile His Pro D-Ala OH 28 Sar Arg Val Tyr Tyr His Pro D-Phe OH 29 SarArg Val  OMTyr Ile His Pro Ala OH 30 Sar Arg Val Tyr OMTh His Pro Ala OH31 Me2G Arg Val Tyr Val His Pro Ala OH 32 Me2G Arg Val Tyr Ile His ProAla OH 33 NMAla Arg Val Tyr Val His Pro Ala OH 34 NMAla Arg Val Tyr IleHis Pro Ala OH 35 Sar Arg Val Tyr Ile His Pro NVA OH 36Cyclo(Arg-Val-Tyr-Ile-His-Pro-Arg-Val-Tyr-Ile-His-Pro-Arg-Val-Tyr-Ile-His-Pro) 37Cyclo(Phe-Val-Tyr-Ile-His-Pro-Phe-Val-Tyr-Ile-His-Pro-Phe-Val-Tyr-Ile-His-Pro) 38 Cyclo(Val-Tyr-Ile-His-Pro-Phe) 40 Sar ArgVal Tyr Glu His Pro NH2 41 Sar Arg Val Tyr Phe His Pro NH2 42 Sar ArgVal Tyr His His Pro NH2 43 Sar Arg Val Tyr Lys His Pro NH2 44 NMAla ArgVal Tyr Ile His Pro D-Ala OH 45 Sar Arg Val Tyr Arg His Pro NH2 46 NMAlaArg Val Tyr Ile His Pro D-Ala OH 47 NM-D- Arg Val Tyr Ile His Pro Ala OHAla 48 Sar Arg Val Phe Ile His Pro D-Ala OH 49 Sar Lys Val Tyr Ile HisPro D-Ala OH 50 Sar Arg Val Tyr Ile Lys Pro D-Ala OH 51 Sar Arg Val TyrIle His D-Pro NH2 52 Sar Arg Val Tyr Tyr His Pro OH 53 Sar Arg Val TyrD-Ala His Pro NH2 54 Sar Arg Val Tyr Val His Pro OH 55 Sar Arg Val TyrVal His NH2 56 Asp Arg Val Tyr Ile His Pro Gly OH 57 Asp Arg Val Tyr TyrHis Pro NH2 58 D-Asp Arg Val Tyr Tyr His Pro NH2 59 Glu Arg Val Tyr TyrHis Pro NH2 60 D-Glu Arg Val Tyr Tyr His Pro NH2 61 N-Me- Arg Val TyrTyr His Pro NH2 Asp 62 N-Me- Arg Val Tyr Tyr His Pro NH2 Glu 63 Asp ArgVal Tyr Phe His Pro NH2 64 Glu Arg Val Tyr Phe His Pro NH2 65 N-Me- ArgVal Tyr Phe His Pro NH2 Asp 66 N-Me- Arg Val Tyr Phe His Pro NH2 Glu 67Sar Arg Val AAA Ile His Pro D-Ala OH 68 Sar Arg Val BBB Ile His ProD-Ala OH 69 Sar Arg Val CCC Ile His Pro D-Ala OH 70 Sar Arg Val DDD IleHis Pro D-Ala OH 71 Sar Arg Val EEE Ile His Pro D-Ala OH 72 Sar Arg ValFFF Ile His Pro D-Ala OH 73 Sar Arg Val GGG Ile His Pro D-Ala OH 74 SarArg Val HHH Ile His Pro D-Ala OH 75 Sar Arg Val III Ile His Pro D-Ala OH76 Sar Arg Val JJJ Ile His Pro D-Ala OH 77 Sar Arg Val AAA OMTh His ProAla OH 78 Sar Arg Val CCC OMTh His Pro Ala OH 79 Sar Arg Val III OMThHis Pro Ala OH 80 MMM Arg Val Tyr Ile His Pro D-Ala OH 81 NNN Arg ValTyr Ile His Pro D-Ala OH 82 NM-D- Arg Val Tyr Ile His Pro D-Ala OH Ala83 Sar Arg Val Tyr NVA His Pro D-Ala OH 84 Sar Arg Val Tyr OMTh His ProD-Ala OH 85 Sar Arg Val OOO Ile His Pro D-Ala OH The definition of theamino acid or its analogues, please refer to the table of abbreviation.Determining GPCR Activity

The compounds of the preferred embodiments are agonists ofβ-arrestin/GRK-mediated signal transduction via the AT1 angiotensinreceptor. The ability of the compounds to effect G protein-mediatedsignaling may be measured using any assay known in the art used todetect G protein-mediated signaling or GPCR activity, or the absence ofsuch signaling/activity. “GPCR activity” refers to the ability of a GPCRto transduce a signal. Such activity can be measured, e.g., in aheterologous cell, by coupling a GPCR (or a chimeric GPCR) to aG-protein and a downstream effector such as PLC or adenylate cyclase,and measuring increases in intracellular calcium (see, e.g., Offermans &Simon, J. Biol. Chem. 270:15175 15180 (1995)). Receptor activity can beeffectively measured by recording ligand-induced changes in [Ca²⁺]_(i)using fluorescent Ca²⁺-indicator dyes and fluorometric imaging. A“natural ligand-induced activity” as used herein, refers to activationof the GPCR by a natural ligand of the GPCR. Activity can be assessedusing any number of endpoints to measure the GPCR activity. For example,activity of a GPCR may be assessed using an assay such as calciummobilization, e.g., an Aequorin luminescence assay.

Generally, assays for testing compounds that modulate GPCR-mediatedsignal transduction include the determination of any parameter that isindirectly or directly under the influence of a GPCR, e.g., afunctional, physical, or chemical effect. It includes ligand binding,changes in ion flux, membrane potential, current flow, transcription,G-protein binding, gene amplification, expression in cancer cells, GPCRphosphorylation or dephosphorylation, signal transduction,receptor-ligand interactions, second messenger concentrations (e.g.,cAMP, cGMP, IP₃, DAG, or intracellular Ca²⁺), in vitro, in vivo, and exvivo and also includes other physiologic effects such as increases ordecreases of neurotransmitter or hormone release; or increases in thesynthesis of particular compounds, e.g., triglycerides. Such parameterscan be measured by any means known to those skilled in the art, e.g.,changes in spectroscopic characteristics (e.g., fluorescence,absorbance, refractive index), hydrodynamic (e.g., shape),chromatographic, or solubility properties, patch clamping,voltage-sensitive dyes, whole cell currents, radioisotope efflux,inducible markers, transcriptional activation of GPCRs; ligand bindingassays; voltage, membrane potential and conductance changes; ion fluxassays; changes in intracellular second messengers such as cAMP andinositol triphosphate (IP₃); changes in intracellular calcium levels;neurotransmitter release, and the like.

When a G protein receptor becomes active, it binds to a G protein (e.g.,Gq, Gs, Gi, Go) and stimulates the binding of GTP to the G protein. TheG protein then acts as a GTPase and slowly hydrolyzes the GTP to GDP,whereby the receptor, under normal conditions, becomes deactivated. Gprotein-mediated signaling or GPCR activity may be measured using assaysystems that are capable of detecting and/or measuring GTP bindingand/or hydrolysis of GTP to GDP.

Gs stimulates the enzyme adenylyl cyclase. Gi (and Go), on the otherhand, inhibit this enzyme. Adenylyl cyclase catalyzes the conversion ofATP to cAMP. Thus, constitutively activated GPCRs that couple the Gsprotein are associated with increased cellular levels of cAMP. On theother hand, activated GPCRs that couple the Gi (or Go) protein areassociated with decreased cellular levels of cAMP. Thus, assays thatdetect cAMP can be utilized to determine if a candidate compound is,e.g., an inverse agonist to the receptor (i.e., such a compound woulddecrease the levels of cAMP). A variety of approaches known in the artfor measuring cAMP can be utilized; a most preferred approach reliesupon the use of anti-cAMP antibodies in an ELISA-based format. Anothertype of assay that can be utilized is a whole cell second messengerreporter system assay. Promoters on genes drive the expression of theproteins that a particular gene encodes. Cyclic AMP drives geneexpression by promoting the binding of a cAMP-responsive DNA bindingprotein or transcription factor (CREB) which then binds to the promoterat specific sites called cAMP response elements and drives theexpression of the gene. Reporter systems can be constructed which have apromoter containing multiple cAMP response elements before the reportergene, e.g., β-galactosidase or luciferase. Thus, a constitutivelyactivated Gs-linked receptor causes the accumulation of cAMP that thenactivates the gene and expression of the reporter protein. The reporterprotein such as β-galactosidase or luciferase can then be detected usingstandard biochemical assays.

Gq and Go are associated with activation of the enzyme phospholipase C,which in turn hydrolyzes the phospholipid PIP2, releasing twointracellular messengers: diacycloglycerol (DAG) and inistol1,4,5-triphoisphate (IP3). Increased accumulation of IP3 is associatedwith activation of Gq- and Go-associated receptors. Assays that detectIP3 accumulation can be utilized to determine if a candidate compoundis, e.g., an inverse agonist to a Gq- or Go-associated receptor (i.e.,such a compound would decrease the levels of IP3). Gq-dependentreceptors can also be examined using an AP1 reporter assay in thatGq-dependent phospholipase C causes activation of genes containing AP1elements.

Samples or assays comprising GPCRs that are treated with a potentialactivator, inhibitor, or modulator are compared to control sampleswithout the inhibitor, activator, or modulator to examine the extent ofinhibition. Control samples (untreated with inhibitors) are assigned arelative GPCR activity value of 100%. Inhibition of a GPCR is achievedwhen the GPCR activity value relative to the control is about 80%,preferably 50%, more preferably 25%. Activation of a GPCR is achievedwhen the GPCR activity value relative to the control (untreated withactivators) is 110%, more preferably 150%, more preferably 200-500%(i.e., two to five fold higher relative to the control), more preferably1000-3000% or higher.

The effects of the compounds upon the function of the GPCR polypeptidescan be measured by examining any of the parameters described above. Anysuitable physiological change that affects GPCR activity can be used toassess the influence of a compound on the GPCRs and naturalligand-mediated GPCR activity. When the functional consequences aredetermined using intact cells or animals, one can also measure a varietyof effects such as transmitter release, hormone release, transcriptionalchanges to both known and uncharacterized genetic markers (e.g.,northern blots), changes in cell metabolism such as cell growth or pHchanges, and changes in intracellular second messengers such as Ca²⁺,IP3 or cAMP.

For a general review of GPCR signal transduction and methods of assayingsignal transduction, see, e.g., Methods in Enzymology, vols. 237 and 238(1994) and volume 96 (1983); Bourne et al., Nature 10:349:117 27 (1991);Bourne et al., Nature 348:125 32 (1990); Pitcher et al., Annu. Rev.Biochem. 67:653 92 (1998).

Modulators of GPCR activity are tested using GPCR polypeptides asdescribed above, either recombinant or naturally occurring. The proteincan be isolated, expressed in a cell, expressed in a membrane derivedfrom a cell, expressed in tissue or in an animal. For example,adipocytes, cells of the immune system, transformed cells, or membranescan be used to test the GPCR polypeptides described above. Modulation istested using one of the in vitro or in vivo assays described herein.Signal transduction can also be examined in vitro with soluble or solidstate reactions, using a chimeric molecule such as an extracellulardomain of a receptor covalently linked to a heterologous signaltransduction domain, or a heterologous extracellular domain covalentlylinked to the transmembrane and or cytoplasmic domain of a receptor.Furthermore, ligand-binding domains of the protein of interest can beused in vitro in soluble or solid state reactions to assay for ligandbinding.

Ligand binding to a GPCR, a domain, or chimeric protein can be tested ina number of formats. Binding can be performed in solution, in a bilayermembrane, attached to a solid phase, in a lipid monolayer, or invesicles. Typically, in an assay of the invention, the binding of thenatural ligand to its receptor is measured in the presence of acandidate modulator. Alternatively, the binding of the candidatemodulator may be measured in the presence of the natural ligand. Often,competitive assays that measure the ability of a compound to competewith binding of the natural ligand to the receptor are used. Binding canbe tested by measuring, e.g., changes in spectroscopic characteristics(e.g., fluorescence, absorbance, refractive index), hydrodynamic (e.g.,shape) changes, or changes in chromatographic or solubility properties.

Receptor-G-protein interactions can also be used to assay formodulators. For example, in the absence of GTP, binding of an activatorsuch as the natural ligand will lead to the formation of a tight complexof a G protein (all three subunits) with the receptor. This complex canbe detected in a variety of ways, as noted above. Such an assay can bemodified to search for inhibitors. For example, the ligand can be addedto the receptor and G protein in the absence of GTP to form a tightcomplex Inhibitors or antagonists may be identified by looking atdissociation of the receptor-G protein complex. In the presence of GTP,release of the α subunit of the G protein from the other two G proteinsubunits serves as a criterion of activation.

An activated or inhibited G-protein will in turn alter the properties ofdownstream effectors such as proteins, enzymes, and channels. Theclassic examples are the activation of cGMP phosphodiesterase bytransducin in the visual system, adenylate cyclase by the stimulatoryG-protein, phospholipase C by Gq and other cognate G proteins, andmodulation of diverse channels by Gi and other G proteins. Downstreamconsequences such as generation of diacyl glycerol and IP3 byphospholipase C, and in turn, for calcium mobilization, e.g., by IP3(further discussed below) can also be examined Thus, modulators can beevaluated for the ability to stimulate or inhibit ligand-mediateddownstream effects. Candidate modulators may be assessed for the abilityto inhibit calcium mobilization induced by nicotinic acid or a relatedcompound that activates the receptor.

In other examples, the ability of a compound to inhibit GPCR activitycan be determined using downstream assays such as measuring lipolysis inadipocytes, release of free fatty acids from adipose tissue, andlipoprotein lipase activity. This may be accomplished, for example,using a competition assay in which varying amounts of a compound areincubated with a GPCR.

Modulators may therefore also be identified using assays involvingβ-arrestin recruitment. β-arrestin serves as a regulatory protein thatis distributed throughout the cytoplasm in unactivated cells. Ligandbinding to an appropriate GPCR is associated with redistribution ofβ-arrestin from the cytoplasm to the cell surface, where it associateswith the GPCR. Thus, receptor activation and the effect of candidatemodulators on ligand-induced receptor activation, can be assessed bymonitoring β-arrestin recruitment to the cell surface. This isfrequently performed by transfecting a labeled β-arrestin fusion protein(e.g., β-arrestin-green fluorescent protein (GFP)) into cells andmonitoring its distribution using confocal microscopy (see, e.g.,Groarke et al., J. Biol. Chem. 274(33):23263 69 (1999)).

Receptor internalization assays may also be used to assess receptorfunction. Upon ligand binding, the G-protein coupled receptor—ligandcomplex is internalized from the plasma membrane by a clathrin-coatedvesicular endocytic process; internalization motifs on the receptorsbind to adaptor protein complexes and mediate the recruitment of theactivated receptors into clathrin-coated pits and vesicles. Because onlyactivated receptors are internalized, it is possible to detectligand-receptor binding by determining the amount of internalizedreceptor. In one assay format, cells are transiently transfected withradiolabeled receptor and incubated for an appropriate period of time toallow for ligand binding and receptor internalization. Thereafter,surface-bound radioactivity is removed by washing with an acid solution,the cells are solubilized, and the amount of internalized radioactivityis calculated as a percentage of ligand binding. See, e.g., Vrecl etal., Mol. Endocrinol. 12:1818 29 (1988) and Conway et al., J. CellPhysiol. 189(3):341 55 (2001). In addition, receptor internalizationapproaches have allowed real-time optical measurements of GPCRinteractions with other cellular components in living cells (see, e.g.,Barak et al., Mol. Pharmacol. 51 (2)177 84 (1997)). Modulators may beidentified by comparing receptor internalization levels in control cellsand cells contacted with candidate compounds.

Another technology that can be used to evaluate GPCR-proteininteractions in living cells involves bioluminescence resonance energytransfer (BRET). A detailed discussion regarding BRET can be found inKroeger et al., J. Biol. Chem., 276 (16):12736 43 (2001).

Receptor-stimulated guanosine 5′-O-(γ-Thio)-Triphosphate ([³⁵S]GTPγS)binding to G-proteins may also be used as an assay for evaluatingmodulators of GPCRs. [³⁵S]GTPγS is a radiolabeled GTP analog that has ahigh affinity for all types of G-proteins, is available with a highspecific activity and, although unstable in the unbound form, is nothydrolyzed when bound to the G-protein. Thus, it is possible toquantitatively assess ligand-bound receptor by comparing stimulatedversus unstimulated [³⁵S]GTPγS binding utilizing, for example, a liquidscintillation counter Inhibitors of the receptor-ligand interactionswould result in decreased [³⁵S]GTPγS binding. Descriptions of [³⁵S]GTPγSbinding assays are provided in Traynor and Nahorski, Mol. Pharmacol.47(4):848 54 (1995) and Bohn et al., Nature 408:720 23 (2000).

The ability of modulators to affect ligand-induced ion flux may also bedetermined. Ion flux may be assessed by determining changes inpolarization (i.e., electrical potential) of the cell or membraneexpressing a GPCR. One means to determine changes in cellularpolarization is by measuring changes in current (thereby measuringchanges in polarization) with voltage-clamp and patch-clamp techniques,e.g., the “cell-attached” mode, the “inside-out” mode, and the “wholecell” mode (see, e.g., Ackerman et al., New Engl. J. Med. 336:1575 1595(1997)). Whole cell currents are conveniently determined using thestandard methodology (see, e.g., Hamil et al., PFlugers. Archiv. 391:85(1981). Other known assays include: radiolabeled ion flux assays andfluorescence assays using voltage-sensitive dyes (see, e.g.,Vestergarrd-Bogind et al., J. Membrane Biol. 88:67 75 (1988); Gonzales &Tsien, Chem. Biol. 4:269 277 (1997); Daniel et al., J. Pharmacol. Meth.25:185 193 (1991); Holevinsky et al., J. Membrane Biology 137:59 70(1994)). Generally, the compounds to be tested are present in the rangefrom 1 pM to 100 mM.

Preferred assays for G-protein coupled receptors include cells that areloaded with ion or voltage sensitive dyes to report receptor activity.Assays for determining activity of such receptors can also use knownagonists and antagonists for other G-protein coupled receptors and thenatural ligands disclosed herein as negative or positive controls toassess activity of tested compounds. In assays for identifyingmodulatory compounds (e.g., agonists, antagonists), changes in the levelof ions in the cytoplasm or membrane voltage are monitored using an ionsensitive or membrane voltage fluorescent indicator, respectively. Amongthe ion-sensitive indicators and voltage probes that may be employed arethose disclosed in the Molecular Probes 1997 Catalog. For G-proteincoupled receptors, promiscuous G-proteins such as Gα15 and Gα16 can beused in the assay of choice (Wilkie et al., Proc. Nat'l Acad. Sci. USA88:10049 10053 (1991)). Such promiscuous G-proteins allow coupling of awide range of receptors to signal transduction pathways in heterologouscells.

As noted above, receptor activation by ligand binding typicallyinitiates subsequent intracellular events, e.g., increases in secondmessengers such as IP3, which releases intracellular stores of calciumions. Activation of some G-protein coupled receptors stimulates theformation of inositol triphosphate (IP3) through phospholipaseC-mediated hydrolysis of phosphatidylinositol (Berridge & Irvine, Nature312:315 21 (1984)). IP3 in turn stimulates the release of intracellularcalcium ion stores. Thus, a change in cytoplasmic calcium ion levels, ora change in second messenger levels such as IP3 can be used to assessG-protein coupled receptor function. Cells expressing such G-proteincoupled receptors may exhibit increased cytoplasmic calcium levels as aresult of contribution from both intracellular stores and via activationof ion channels, in which case it may be desirable although notnecessary to conduct such assays in calcium-free buffer, optionallysupplemented with a chelating agent such as EGTA, to distinguishfluorescence response resulting from calcium release from internalstores.

Other assays can involve determining the activity of receptors which,when activated by ligand binding, result in a change in the level ofintracellular cyclic nucleotides, e.g., cAMP or cGMP, by activating orinhibiting downstream effectors such as adenylate cyclase. In oneembodiment, changes in intracellular cAMP or cGMP can be measured usingimmunoassays. The method described in Offermanns & Simon, J. Biol. Chem.270:15175 15180 (1995) may be used to determine the level of cAMP. Also,the method described in Felley-Bosco et al., Am. J. Resp. Cell and Mol.Biol. 11:159 164 (1994) may be used to determine the level of cGMP.Further, an assay kit for measuring cAMP and/or cGMP is described inU.S. Pat. No. 4,115,538, herein incorporated by reference.

In another embodiment, phosphatidyl inositol (PI) hydrolysis can beanalyzed according to U.S. Pat. No. 5,436,128, herein incorporated byreference. Briefly, the assay involves labeling of cells with3H-myoinositol for 48 or more hrs. The labeled cells are treated with acompound for one hour. The treated cells are lysed and extracted inchloroform-methanol-water after which the inositol phosphates areseparated by ion exchange chromatography and quantified by scintillationcounting. Fold stimulation is determined by calculating the ratio ofcounts per minute (cpm) in the presence of agonist to cpm in thepresence of buffer control. Likewise, fold inhibition is determined bycalculating the ratio of cpm in the presence of antagonist to cpm in thepresence of buffer control (which may or may not contain an agonist).

In another embodiment, transcription levels can be measured to assessthe effects of a test compound on ligand-induced signal transduction. Ahost cell containing the protein of interest is contacted with a testcompound in the presence of the natural ligand for a sufficient time toeffect any interactions, and then the level of gene expression ismeasured. The amount of time to effect such interactions may beempirically determined, such as by running a time course and measuringthe level of transcription as a function of time. The amount oftranscription may be measured by using any method known to those ofskill in the art to be suitable. For example, mRNA expression of theprotein of interest may be detected using northern blots or theirpolypeptide products may be identified using immunoassays.Alternatively, transcription based assays using reporter genes may beused as described in U.S. Pat. No. 5,436,128, herein incorporated byreference. The reporter genes can be, e.g., chloramphenicolacetyltransferase, firefly luciferase, bacterial luciferase,β-galactosidase and alkaline phosphatase. Furthermore, the protein ofinterest can be used as an indirect reporter via attachment to a secondreporter such as green fluorescent protein (see, e.g., Mistili &Spector, Nature Biotechnology 15:961 964 (1997)).

The amount of transcription is then compared to the amount oftranscription in either the same cell in the absence of the testcompound, or it may be compared with the amount of transcription in asubstantially identical cell that lacks the protein of interest. Asubstantially identical cell may be derived from the same cells fromwhich the recombinant cell was prepared but which had not been modifiedby introduction of heterologous DNA. Any difference in the amount oftranscription indicates that the test compound has in some manneraltered the activity of the protein of interest.

Samples that are treated with a GPCR antagonist are compared to controlsamples comprising the natural ligand without the test compound toexamine the extent of modulation. Control samples (untreated withactivators or inhibitors) are assigned a relative GPCR activity value of100 Inhibition of a GPCR is achieved when the GPCR activity valuerelative to the control is about 90%, optionally 50%, or optionally 25%.Activation of a GPCR is achieved when the GPCR activity value relativeto the control is 110%, optionally 150%, 200-500%, or 1000-2000%.

Determining β-Arrestin/GRK-Mediated Signal Transduction

The ability of the compounds of the present invention to activateβ-arrestin/GRK-mediated signal transduction via the AT1 angiotensinreceptor may be measured using any assay known in the art used to detectβ-arrestin/GRK-mediated signal transduction via the AT1 angiotensinreceptor, or the absence of such signal transduction. Generally,activated GPCRs become substrates for kinases that phosphorylate theC-terminal tail of the receptor (and possibly other sites as well).Thus, an antagonist will inhibit the transfer of ³²P from gamma-labeledGTP to the receptor, which can be assayed with a scintillation counter.The phosphorylation of the C-terminal tail will promote the binding ofarrestin-like proteins and will interfere with the binding ofG-proteins. The kinase/arrestin pathway plays a key role in thedesensitization of many GPCR receptors.

The proximal event in β-arrestin function mediated by GPCRs isrecruitment to receptors following ligand binding and receptorphosphorylation by GRK's. Thus, measure of β-arrestin recruitment wasused to determine ligand efficacy for β-arrestin function.

Peptides, Derivatives and Mimetics

The terms “peptidyl” and “peptidic” as used throughout the specificationand claims are intended to include active derivatives, variants, and/ormimetics of the peptides according to the present embodiments. Peptidiccompounds are structurally similar bioactive equivalents of the peptidesaccording to the present embodiments. By a “structurally similarbioactive equivalent” is meant a peptidyl compound with structuresufficiently similar to that of an identified bioactive peptide toproduce substantially equivalent therapeutic effects. For example,peptidic compounds derived from the amino acid sequence of the peptide,or having an amino acid sequence backbone of the peptide, are consideredstructurally similar bioactive equivalents of the peptide.

The term “variant” refers to a protein or polypeptide in which one ormore amino acid substitutions, deletions, and/or insertions are presentas compared to the amino acid sequence of an protein or peptide andincludes naturally occurring allelic variants or alternative splicevariants of an protein or peptide. The term “variant” includes thereplacement of one or more amino acids in a peptide sequence with asimilar or homologous amino acid(s) or a dissimilar amino acid(s).Preferred variants include alanine substitutions at one or more of aminoacid positions. Other preferred substitutions include conservativesubstitutions that have little or no effect on the overall net charge,polarity, or hydrophobicity of the protein. Conservative substitutionsare set forth in the table below. According to some embodiments, the CKpolypeptides have at least 60%, 65%, 70%, 75%, 80%, 85%, 88%, 95%, 96%,97%, 98% or 99% sequence identity with the amino acid or amino acidanalogue sequences of the preferred embodiments.

Conservative Amino Acid Substitutions

Basic: arginine lysine histidine Acidic: glutamic acid aspartic acidUncharged glutamine Polar: asparagine serine threonine tyrosineNon-Polar: phenylalanine tryptophan cysteine glycine alanine valinepraline methionine leucine isoleucine

The table below sets out another scheme of amino acid substitution:

Original Residue Substitutions Ala Gly; Ser Arg Lys Asn Gln; His Asp GluCys Ser Gln Asn Glu Asp Gly Ala; Pro His Asn; Gln Ile Leu; Val Leu Ile;Val Lys Arg; Gln; Glu Met Leu; Tyr; Ile Phe Met; Leu; Tyr Ser Thr ThrSer Trp Tyr Tyr Trp; Phe Val Ile; Leu

Other variants can consist of less conservative amino acidsubstitutions, such as selecting residues that differ more significantlyin their effect on maintaining (a) the structure of the polypeptidebackbone in the area of the substitution, for example, as a sheet orhelical conformation, (b) the charge or hydrophobicity of the moleculeat the target site, or (c) the bulk of the side chain. The substitutionsthat in general are expected to have a more significant effect onfunction are those in which (a) glycine and/or proline is substituted byanother amino acid or is deleted or inserted; (b) a hydrophilic residue,e.g., seryl or threonyl, is substituted for (or by) a hydrophobicresidue, e.g., leucyl, isoleucyl, phenylalanyl, valyl, or alanyl; (c) acysteine residue is substituted for (or by) any other residue; (d) aresidue having an electropositive side chain, e.g., lysyl, arginyl, orhistidyl, is substituted for (or by) a residue having an electronegativecharge, e.g., glutamyl or aspartyl; or (e) a residue having a bulky sidechain, e.g., phenylalanine, is substituted for (or by) one not havingsuch a side chain, e.g., glycine. Other variants include those designedto either generate a novel glycosylation and/or phosphorylation site(s),or those designed to delete an existing glycosylation and/orphosphorylation site(s). Variants include at least one amino acidsubstitution at a glycosylation site, a proteolytic cleavage site and/ora cysteine residue. Variants also include proteins and peptides withadditional amino acid residues before or after the protein or peptideamino acid sequence on linker peptides. The term “variant” alsoencompasses polypeptides that have the amino acid sequence of theproteins/peptides of the present invention with at least one and up to25 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20) additional amino acidsflanking either the 3′ or 5′ end of the amino acid sequence or both.

The term “variant” also refers to a protein that is at least 60 to 99percent identical (e.g., 60, 65, 70, 75, 80, 85, 90, 95, 98, 99, or100%, inclusive) in its amino acid sequence of the proteins of thepresent invention as determined by standard methods that are commonlyused to compare the similarity in position of the amino acids of twopolypeptides. The degree of similarity or identity between two proteinscan be readily calculated by known methods. Preferred methods todetermine identity are designed to give the largest match between thesequences tested. Methods to determine identity and similarity arecodified in publicly available computer programs. Variants willtypically have one or more (e.g., 2, 3, 4, 5, etc.) amino acidsubstitutions, deletions, and/or insertions as compared with thecomparison protein or peptide, as the case may be.

The compounds of the present invention include compounds having one ofthe general formulas described herein, in addition to derivatives and/ormimetics thereof.

The term “derivative” refers to a chemically modified protein orpolypeptide that has been chemically modified either by naturalprocesses, such as processing and other post-translationalmodifications, but also by chemical modification techniques, as forexample, by addition of one or more polyethylene glycol molecules,sugars, phosphates, and/or other such molecules, where the molecule ormolecules are not naturally attached to wild-type proteins. Derivativesinclude salts. Such chemical modifications are well described in basictexts and in more detailed monographs, as well as in a voluminousresearch literature, and they are well known to those of skill in theart. It will be appreciated that the same type of modification may bepresent in the same or varying degree at several sites in a givenprotein or polypeptide. Also, a given protein or polypeptide may containmany types of modifications. Modifications can occur anywhere in aprotein or polypeptide, including the peptide backbone, the amino acidside-chains, and the amino or carboxyl termini. Modifications include,for example, acetylation, acylation, ADP-ribosylation, amidation,covalent attachment of flavin, covalent attachment of a heme moiety,covalent attachment of a nucleotide or nucleotide derivative, covalentattachment of a lipid or lipid derivative, covalent attachment ofphosphotidylinositol, cross-linking, cyclization, disulfide bondformation, demethylation, formation of covalent cross-links, formationof cysteine, formation of pyroglutamate, formylation,gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation,iodination, methylation, myristoylation, oxidation, proteolyticprocessing, phosphorylation, prenylation, racemization, glycosylation,lipid attachment, sulfation, gamma-carboxylation of glutamic acidresidues, hydroxylation and ADP-ribosylation, selenoylation, sulfation,transfer-RNA mediated addition of amino acids to proteins, such asarginylation, and ubiquitination. See, for instance, Proteins—StructureAnd Molecular Properties, 2nd Ed., T. E. Creighton, W. H. Freeman andCompany, New York (1993) and Wold, F., “Posttranslational ProteinModifications: Perspectives and Prospects,” pgs. 1-12 inPosttranslational Covalent Modification Of Proteins, B. C. Johnson, Ed.,Academic Press, New York (1983); Seifter et al., Meth. Enzymol.182:626-646 (1990) and Rattan et al., “Protein Synthesis:Posttranslational Modifications and Aging,” Ann. N.Y. Acad. Sci. 663:48-62 (1992). The term “derivatives” include chemical modificationsresulting in the protein or polypeptide becoming branched or cyclic,with or without branching. Cyclic, branched and branched circularproteins or polypeptides may result from post-translational naturalprocesses and may be made by entirely synthetic methods, as well.

According to some embodiments, the compounds of the present inventionmay optionally include compounds wherein the N-terminus is derivatizedto a —NRR¹ group; to a —NRC(O)R group; to a —NRC(O)OR group; to a—NRS(O)₂R group; to a —NHC(O)NHR group, where R and R¹ are hydrogen orlower alkyl with the proviso that R and R¹ are not both hydrogen; to asuccinimide group; to a benzyloxycarbonyl-NH—(CBZ—CH—) group; or to abenzyloxycarbonyl-NE-group having from 1 to 3 substituents on the phenylring selected from the group consisting of lower alkyl, lower alkoxy,chloro, and bromo.

According to some embodiments, the compounds of the present inventionmay optionally include compounds wherein the C terminus is derivatizedto —C(O)R² where R² is selected from the group consisting of loweralkoxy, and —NR³R⁴ where R³ and R⁴ are independently selected from thegroup consisting of hydrogen and lower alkyl.

The term “peptide mimetic” or “mimetic” refers to biologically activecompounds that mimic the biological activity of a peptide or a proteinbut are no longer peptidic in chemical nature, that is, they no longercontain any peptide bonds (that is, amide bonds between amino acids).Here, the term peptide mimetic is used in a broader sense to includemolecules that are no longer completely peptidic in nature, such aspseudo-peptides, semi-peptides and peptoids. Examples of peptidemimetics in this broader sense (where part of a peptide is replaced by astructure lacking peptide bonds) are described below. Whether completelyor partially non-peptide, peptide mimetics according to the embodimentsprovide a spatial arrangement of reactive chemical moieties that closelyresemble the three-dimensional arrangement of active groups in thepeptide on which the peptide mimetic is based. As a result of thissimilar active-site geometry, the peptide mimetic has effects onbiological systems that are similar to the biological activity of thepeptide.

The peptides and peptide mimetics included in the compositions of theinvention are 6 to 25, 6 to 20, 6 to 15, 6 to 10, 6 to 9, 6 to 8, 7 to25, 7 to 20, 7 to 15, 7 to 12, 7 to 10, 7 to 9, 7 to 8, 8 to 25, 8 to20, 8 to 15, 8 to 12, 8 to 10, 8 to 9, 9 to 25, 9 to 20, 9 to 18, 9 to15, 9 to 14, 9 to 12, 10 to 25, 10 to 20, 10 to 15, 10 to 14, 10 to 12,12 to 25 or 12 to 20 amino acids or amino acid analogues in length.

The peptide mimetics of the embodiments are preferably substantiallysimilar in both three-dimensional shape and biological activity to thepeptides described herein. According to some embodiments, peptidemimetics of the present invention have protective groups at one or bothends of the compounds of the inventions, and/or replacement of one ormore peptide bonds with non-peptide bonds. Such modifications may renderthe compounds less susceptible to proteolytic cleavage than the compounditself. For instance, one or more peptide bonds can be replaced with analternative type of covalent bond (e.g., a carbon-carbon bond or an acylbond). Peptide mimetics can also incorporate amino-terminal or carboxylterminal blocking groups such as t-butyloxycarbonyl, acetyl, alkyl,succinyl, methoxysuccinyl, suberyl, adipyl, azelayl, dansyl,benzyloxycarbonyl, fluorenylmethoxycarbonyl, methoxyazelayl,methoxyadipyl, methoxysuberyl, and 2,4,-dinitrophenyl, thereby renderingthe mimetic less susceptible to proteolysis. Non-peptide bonds andcarboxyl- or amino-terminal blocking groups can be used singly or incombination to render the mimetic less susceptible to proteolysis thanthe corresponding peptide/compound. Additionally, substitution ofD-amino acids for the normal L-stereoisomer can be effected, e.g. toincrease the half-life of the molecule.

Thus, according to some embodiments, the compounds of the presentinvention may optionally include a pseudopeptide bond wherein one ormore of the peptidyl [—C(O)NR—] linkages (bonds) have been replaced by anon-peptidyl linkage such as —CH₂—NH—, —CH₂—S—, —CH₂—SO—, —CH₂—SO₂—,—NH—CO—, or —CH═CH— replacing a peptide bond (—CO—NH—). According tosome embodiments, the compounds of the present invention may optionallyinclude a pseudopeptide bond wherein one or more of the peptidyl[—C(O)NR—] linkages (bonds) have been replaced by a non-peptidyl linkagesuch as a —CH₂-carbamate linkage [—CH₂—OC(O)NR—]; a phosphonate linkage;a —CH₂-sulfonamide [—CH₂—S(O)₂NR—] linkage; a urea [—NHC(O)NH—] linkage;a —CH₂-secondary amine linkage; or an alkylated peptidyl linkage[—C(O)NR⁶— where R⁶ is lower alkyl]. Preferred mimetics have from zeroto all of the —C(O)NH— linkages of the of the present invention replacedby a pseudopeptide.

Examples of methods of structurally modifying a peptide known in the artto create a peptide mimetic include the inversion of backbone chiralcenters leading to D-amino acid residue structures that may,particularly at the N-terminus, lead to enhanced stability forproteolytical degradation without adversely affecting activity. Anexample is given in the paper “Tritriated D-ala¹-Peptide T Binding”,Smith C. S. et al., Drug Development Res., 15, pp. 371-379 (1988). Asecond method is altering cyclic structure for stability, such as N to Cinterchain imides and lactames (Ede et al. in Smith and Rivier (Eds.)“Peptides: Chemistry and Biology”, Escom, Leiden (1991), pp. 268-270).An example of this is given in conformationally restrictedthymopentin-like compounds, such as those disclosed in U.S. Pat. No.4,457,489 (1985), Goldstein, G. et al., the disclosure of which isincorporated by reference herein in its entirety. A third method is tosubstitute peptide bonds in the peptide by pseudopeptide bonds thatconfer resistance to proteolysis. The synthesis of peptides containingpseudopeptide bonds such as —CH₂—NH—, —CH₂—S—, —CH₂—SO—, —CH₂—SO₂—,—NH—CO— or —CH═CH— is performed either by solution methods or in acombined procedure with solid-phase synthesis using standard methods oforganic chemistry. Thus, for example, the introduction of the —CH₂—NH—bond is accomplished by preparing in solution the aldehydeFmoc-NH—CHR—CHO according to the technique described by FEHRENTZ andCASTRO (Synthesis, 676-678, 1983) and condensing it with the growingpeptide chain, either on a solid phase according to the techniquedescribed by SASAKI and COY (Peptides, 8, 119-121, 1988), or insolution.

Pharmaceutical Compositions/Formulations

Pharmaceutical compositions for use in the present invention can beformulated by standard techniques using one or more physiologicallyacceptable carriers or excipients. In a preferred embodiment of theinvention, the formulations may contain a buffer and/or a preservative.The compounds and their physiologically acceptable salts and solvatescan be formulated for administration by any suitable route, includingvia inhalation, topically, nasally, orally, parenterally (e.g.,intravenously, intraperitoneally, intravesically or intrathecally) orrectally in a vehicle comprising one or more pharmaceutically acceptablecarriers, the proportion of which is determined by the solubility andchemical nature of the peptide, chosen route of administration andstandard biological practice.

According to some embodiments, pharmaceutical compositions are providedcomprising effective amounts of one or more compound(s) of the presentinvention together with, for example, pharmaceutically acceptablediluents, preservatives, solubilizers, emulsifiers, adjuvants and/orother carriers. Such compositions include diluents of various buffercontent (e.g., TRIS or other amines, carbonates, phosphates, aminoacids, for example, glycinamide hydrochloride (especially in thephysiological pH range), N-glycylglycine, sodium or potassium phosphate(dibasic, tribasic), etc. or TRIS-HCl or acetate), pH and ionicstrength; additives such as detergents and solubilizing agents (e.g.,surfactants such as Pluronics, Tween 20, Tween 80 (Polysorbate 80),Cremophor, polyols such as polyethylene glycol, propylene glycol, etc.),anti-oxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives(e.g., Thimersol, benzyl alcohol, parabens, etc.) and bulking substances(e.g., sugars such as sucrose, lactose, mannitol, polymers such aspolyvinylpyrrolidones or dextran, etc.); and/or incorporation of thematerial into particulate preparations of polymeric compounds such aspolylactic acid, polyglycolic acid, etc. or into liposomes. Hyaluronicacid may also be used. Such compositions can be employed to influencethe physical state, stability, rate of in vivo release, and rate of invivo clearance of a compound of the present invention. See, e.g.,Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack PublishingCo., Easton, Pa. 18042) pages 1435-1712 which are herein incorporated byreference. The compositions can, for example, be prepared in liquidform, or can be in dried powder, such as lyophilized form. Particularmethods of administering such compositions are described infra.

Where a buffer is to be included in the formulations of the invention,the buffer is selected from the group consisting of sodium acetate,sodium carbonate, citrate, glycylglycine, histidine, glycine, lysine,arginin, sodium dihydrogen phosphate, disodium hydrogen phosphate,sodium phosphate, and tris(hydroxymethyl)-aminomethan, or mixturesthereof. Each one of these specific buffers constitutes an alternativeembodiment of the invention. In a preferred embodiment of the inventionthe buffer is glycylglycine, sodium dihydrogen phosphate, disodiumhydrogen phosphate, sodium phosphate or mixtures thereof.

Where a pharmaceutically acceptable preservative is to be included inthe formulations of the invention, the preservative is selected from thegroup consisting of phenol, m-cresol, methyl p-hydroxybenzoate, propylp-hydroxybenzoate, 2-phenoxyethanol, butyl p-hydroxybenzoate,2-phenylethanol, benzyl alcohol, chlorobutanol, and thiomerosal, ormixtures thereof. Each one of these specific preservatives constitutesan alternative embodiment of the invention. In a preferred embodiment ofthe invention the preservative is phenol or m-cresol.

In a further embodiment of the invention the preservative is present ina concentration from about 0.1 mg/ml to about 50 mg/ml, more preferablyin a concentration from about 0.1 mg/ml to about 25 mg/ml, and mostpreferably in a concentration from about 0.1 mg/ml to about 10 mg/ml.

The use of a preservative in pharmaceutical compositions is well-knownto the skilled person. For convenience reference is made to Remington:The Science and Practice of Pharmacy, 19th edition, 1995.

In a further embodiment of the invention the formulation may furthercomprise a chelating agent where the chelating agent may be selectedfrom salts of ethlenediaminetetraacetic acid (EDTA), citric acid, andaspartic acid, and mixtures thereof. Each one of these specificchelating agents constitutes an alternative embodiment of the invention.

In a further embodiment of the invention the chelating agent is presentin a concentration from 0.1 mg/ml to 5 mg/ml. In a further embodiment ofthe invention the chelating agent is present in a concentration from 0.1mg/ml to 2 mg/ml. In a further embodiment of the invention the chelatingagent is present in a concentration from 2 mg/ml to 5 mg/ml.

The use of a chelating agent in pharmaceutical compositions iswell-known to the skilled person. For convenience reference is made toRemington: The Science and Practice of Pharmacy, 19th edition, 1995.

In a further embodiment of the invention the formulation may furthercomprise a stabiliser selected from the group of high molecular weightpolymers or low molecular compounds where such stabilizers include, butare not limited to, polyethylene glycol (e.g. PEG 3350),polyvinylalcohol (PVA), polyvinylpyrrolidone, carboxymethylcellulose,different salts (e.g. sodium chloride), L-glycine, L-histidine,imidazole, arginine, lysine, isoleucine, aspartic acid, tryptophan,threonine and mixtures thereof. Each one of these specific stabilizersconstitutes an alternative embodiment of the invention. In a preferredembodiment of the invention the stabiliser is selected from the groupconsisting of L-histidine, imidazole and arginine.

In a further embodiment of the invention the high molecular weightpolymer is present in a concentration from 0.1 mg/ml to 50 mg/ml. In afurther embodiment of the invention the high molecular weight polymer ispresent in a concentration from 0.1 mg/ml to 5 mg/ml. In a furtherembodiment of the invention the high molecular weight polymer is presentin a concentration from 5 mg/ml to 10 mg/ml. In a further embodiment ofthe invention the high molecular weight polymer is present in aconcentration from 10 mg/ml to 20 mg/ml. In a further embodiment of theinvention the high molecular weight polymer is present in aconcentration from 20 mg/ml to 30 mg/ml. In a further embodiment of theinvention the high molecular weight polymer is present in aconcentration from 30 mg/ml to 50 mg/ml.

In a further embodiment of the invention the low molecular weightcompound is present in a concentration from 0.1 mg/ml to 50 mg/ml. In afurther embodiment of the invention the low molecular weight compound ispresent in a concentration from 0.1 mg/ml to 5 mg/ml. In a furtherembodiment of the invention the low molecular weight compound is presentin a concentration from 5 mg/ml to 10 mg/ml. In a further embodiment ofthe invention the low molecular weight compound is present in aconcentration from 10 mg/ml to 20 mg/ml. In a further embodiment of theinvention the low molecular weight compound is present in aconcentration from 20 mg/ml to 30 mg/ml. In a further embodiment of theinvention the low molecular weight compound is present in aconcentration from 30 mg/ml to 50 mg/ml.

The use of a stabilizer in pharmaceutical compositions is well-known tothe skilled person. For convenience reference is made to Remington: TheScience and Practice of Pharmacy, 19th edition, 1995.

In a further embodiment of the invention the formulation of theinvention may further comprise a surfactant where a surfactant may beselected from a detergent, ethoxylated castor oil, polyglycolyzedglycerides, acetylated monoglycerides, sorbitan fatty acid esters,poloxamers, such as 188 and 407, polyoxyethylene sorbitan fatty acidesters, polyoxyethylene derivatives such as alkylated and alkoxylatedderivatives (tweens, e.g. Tween-20, or Tween-80), monoglycerides orethoxylated derivatives thereof, diglycerides or polyoxyethylenederivatives thereof, glycerol, cholic acid or derivatives thereof,lecithins, alcohols and phospholipids, glycerophospholipids (lecithins,kephalins, phosphatidyl serine), glyceroglycolipids (galactopyransoide),sphingophospholipids (sphingomyelin), and sphingoglycolipids (ceramides,gangliosides), DSS (docusate sodium, docusate calcium, docusatepotassium, SDS (sodium dodecyl sulfate or sodium lauryl sulfate),dipalmitoyl phosphatidic acid, sodium caprylate, bile acids and saltsthereof and glycine or taurine conjugates, ursodeoxycholic acid, sodiumcholate, sodium deoxycholate, sodium taurocholate, sodium glycocholate,N-Hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, anionic(alkyl-aryl-sulphonates) monovalent surfactants, palmitoyllysophosphatidyl-L-serine, lysophospholipids (e.g.1-acyl-sn-glycero-3-phosphate esters of ethanolamine, choline, serine orthreonine), alkyl, alkoxyl (alkyl ester), alkoxy (alkylether)-derivatives of lysophosphatidyl and phosphatidylcholines, e.g.lauroyl and myristoyl derivatives of lysophosphatidylcholine,dipalmitoylphosphatidylcholine, and modifications of the polar headgroup, that is cholines, ethanolamines, phosphatidic acid, serines,threonines, glycerol, inositol, and the positively charged DODAC, DOTMA,DCP, BISHOP, lysophosphatidylserine and lysophosphatidylthreonine,zwitterionic surfactants (e.g.N-alkyl-N,N-dimethylammonio-1-propanesulfonates,3-cholamido-1-propyldimethylammonio-1-propanesulfonate,dodecylphosphocholine, myristoyl lysophosphatidylcholine, hen egglysolecithin), cationic surfactants (quarternary ammonium bases) (e.g.cetyl-trimethylammonium bromide, cetylpyridinium chloride), non-ionicsurfactants, polyethyleneoxide/polypropyleneoxide block copolymers(Pluronics/Tetronics, Triton X-100, Dodecyl β-D-glucopyranoside) orpolymeric surfactants (Tween-40, Tween-80, Brij-35), fusidic acidderivatives—(e.g. sodium tauro-dihydrofusidate etc.), long-chain fattyacids and salts thereof C6-C12 (e.g. oleic acid and caprylic acid),acylcarnitines and derivatives, N_(α)-acylated derivatives of lysine,arginine or histidine, or side-chain acylated derivatives of lysine orarginine, N_(α)-acylated derivatives of dipeptides comprising anycombination of lysine, arginine or histidine and a neutral or acidicamino acid, N_(α)-acylated derivative of a tripeptide comprising anycombination of a neutral amino acid and two charged amino acids, or thesurfactant may be selected from the group of imidazoline derivatives, ormixtures thereof. Each one of these specific surfactants constitutes analternative embodiment of the invention.

The use of a surfactant in pharmaceutical compositions is well-known tothe skilled person. For convenience reference is made to Remington: TheScience and Practice of Pharmacy, 19th edition, 1995.

Pharmaceutically acceptable sweeteners comprise preferably at least oneintense sweetener such as saccharin, sodium or calcium saccharin,aspartame, acesulfame potassium, sodium cyclamate, alitame, adihydrochalcone sweetener, monellin, stevioside or sucralose(4,1′,6′-trichloro-4,1′,6′-trideoxygalactosucrose), preferablysaccharin, sodium or calcium saccharin, and optionally a bulk sweetenersuch as sorbitol, mannitol, fructose, sucrose, maltose, isomalt,glucose, hydrogenated glucose syrup, xylitol, caramel or honey.

Intense sweeteners are conveniently employed in low concentrations. Forexample, in the case of sodium saccharin, the concentration may rangefrom 0.04% to 0.1% (w/v) based on the total volume of the finalformulation, and preferably is about 0.06% in the low-dosageformulations and about 0.08% in the high-dosage ones. The bulk sweetenercan effectively be used in larger quantities ranging from about 10% toabout 35%, preferably from about 10% to 15% (w/v).

The formulations of the invention may be prepared by conventionaltechniques, e.g. as described in Remington's Pharmaceutical Sciences,1985 or in Remington: The Science and Practice of Pharmacy, 19thedition, 1995, where such conventional techniques of the pharmaceuticalindustry involve dissolving and mixing the ingredients as appropriate togive the desired end product.

The phrase “pharmaceutically acceptable” or “therapeutically acceptable”refers to molecular entities and compositions that are physiologicallytolerable and preferably do not typically produce an allergic or similaruntoward reaction, such as gastric upset, dizziness and the like, whenadministered to a human. Preferably, as used herein, the term“pharmaceutically acceptable” means approved by a regulatory agency ofthe Federal or a State government or listed in the U.S. Pharmacopeia orother generally recognized pharmacopeia (e.g., Remington'sPharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985))for use in animals, and more particularly in humans.

Administration of the compounds of the present invention may be carriedout using any method known in the art. For example, administration maybe transdermal, parenteral, intravenous, intra-arterial, subcutaneous,intramuscular, intracranial, intraorbital, ophthalmic, intraventricular,intracapsular, intraspinal, intracisternal, intraperitoneal,intracerebroventricular, intrathecal, intranasal, aerosol, bysuppositories, or oral administration. Preferably, a pharmaceuticalcomposition of the present invention can be for administration forinjection, or for oral, pulmonary, nasal, transdermal, ocularadministration.

For oral administration, the peptide or a therapeutically acceptablesalt thereof can be formulated in unit dosage forms such as capsules ortablets. The tablets or capsules may be prepared by conventional meanswith pharmaceutically acceptable excipients, including binding agents,for example, pregelatinised maize starch, polyvinylpyrrolidone, orhydroxypropyl methylcellulose; fillers, for example, lactose,microcrystalline cellulose, or calcium hydrogen phosphate; lubricants,for example, magnesium stearate, talc, or silica; disintegrants, forexample, potato starch or sodium starch glycolate; or wetting agents,for example, sodium lauryl sulphate. Tablets can be coated by methodswell known in the art. Liquid preparations for oral administration cantake the form of, for example, solutions, syrups, or suspensions, orthey can be presented as a dry product for constitution with water orother suitable vehicle before use. Such liquid preparations can beprepared by conventional means with pharmaceutically acceptableadditives, for example, suspending agents, for example, sorbitol syrup,cellulose derivatives, or hydrogenated edible fats; emulsifying agents,for example, lecithin or acacia; non-aqueous vehicles, for example,almond oil, oily esters, ethyl alcohol, or fractionated vegetable oils;and preservatives, for example, methyl or propyl-p-hydroxybenzoates orsorbic acid. The preparations can also contain buffer salts, flavoring,coloring, and/or sweetening agents as appropriate. If desired,preparations for oral administration can be suitably formulated to givecontrolled release of the active compound.

For topical administration, the peptide can be formulated in apharmaceutically acceptable vehicle containing 0.1 to 10 percent,preferably 0.5 to 5 percent, of the active compound(s). Suchformulations can be in the form of a cream, lotion, sublingual tablet,aerosols and/or emulsions and can be included in a transdermal or buccalpatch of the matrix or reservoir type as are conventional in the art forthis purpose.

For parenteral administration, the compounds of the present inventionare administered by either intravenous, subcutaneous, or intramuscularinjection, in compositions with pharmaceutically acceptable vehicles orcarriers. The compounds can be formulated for parenteral administrationby injection, for example, by bolus injection or continuous infusion.Formulations for injection can be presented in unit dosage form, forexample, in ampoules or in multi-dose containers, with an addedpreservative. The compositions can take such forms as suspensions,solutions, or emulsions in oily or aqueous vehicles, and can containformulatory agents, for example, suspending, stabilizing, and/ordispersing agents. Alternatively, the active ingredient can be in powderform for constitution with a suitable vehicle, for example, sterilepyrogen-free water, before use.

For administration by injection, it is preferred to use the compound(s)in solution in a sterile aqueous vehicle which may also contain othersolutes such as buffers or preservatives as well as sufficientquantities of pharmaceutically acceptable salts or of glucose to makethe solution isotonic. In some embodiments, the pharmaceuticalcompositions of the present invention may be formulated with apharmaceutically acceptable carrier to provide sterile solutions orsuspensions for injectable administration. In particular, injectablescan be prepared in conventional forms, either as liquid solutions orsuspensions, solid forms suitable for solution or suspensions in liquidprior to injection or as emulsions. Suitable excipients are, forexample, water, saline, dextrose, mannitol, lactose, lecithin, albumin,sodium glutamate, cysteine hydrochloride, or the like. In addition, ifdesired, the injectable pharmaceutical compositions may contain minoramounts of nontoxic auxiliary substances, such as wetting agents, pHbuffering agents, and the like. If desired, absorption enhancingpreparations (e.g., liposomes) may be utilized. Suitable pharmaceuticalcarriers are described in “Remington's pharmaceutical Sciences” by E. W.Martin.

For administration by inhalation, the compounds may be convenientlydelivered in the form of an aerosol spray presentation from pressurizedpacks or a nebulizer, with the use of a suitable propellant, forexample, dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide, or other suitable gas. In thecase of a pressurized aerosol, the dosage unit can be determined byproviding a valve to deliver a metered amount. Capsules and cartridgesof, for example, gelatin for use in an inhaler or insufflator can beformulated containing a powder mix of the compound and a suitable powderbase, for example, lactose or starch. For intranasal administration thecompounds of the invention may be used, for example, as a liquid spray,as a powder or in the form of drops.

The compounds can also be formulated in rectal compositions, forexample, suppositories or retention enemas, for example, containingconventional suppository bases, for example, cocoa butter or otherglycerides.

Furthermore, the compounds can be formulated as a depot preparation.Such long-acting formulations can be administered by implantation (forexample, subcutaneously or intramuscularly) or by intramuscularinjection. Thus, for example, the compounds can be formulated withsuitable polymeric or hydrophobic materials (for example as an emulsionin an acceptable oil) or ion exchange resins, or as sparingly solublederivatives, for example, as a sparingly soluble salt.

The compositions can, if desired, be presented in a pack or dispenserdevice that can contain one or more unit dosage forms containing theactive ingredient. The pack can, for example, comprise metal or plasticfoil, for example, a blister pack. The pack or dispenser device can beaccompanied by instructions for administration.

Dosages

The compounds of the present invention may be administered to a patientat therapeutically effective doses to prevent, treat, or controldiseases and disorders mediated, in whole or in part, by a GPCR-ligandinteraction of the present invention. Pharmaceutical compositionscomprising one or more of compounds of the present invention may beadministered to a patient in an amount sufficient to elicit an effectiveprotective or therapeutic response in the patient. An amount adequate toaccomplish this is defined as “therapeutically effective dose.” The dosewill be determined by the efficacy of the particular compound employedand the condition of the subject, as well as the body weight or surfacearea of the area to be treated. The size of the dose also will bedetermined by the existence, nature, and extent of any adverse effectsthat accompany the administration of a particular compound or vector ina particular subject.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, for example, by determining the LD50 (the dose lethal to 50% ofthe population) and the ED50 (the dose therapeutically effective in 50%of the population). The dose ratio between toxic and therapeutic effectsis the therapeutic index and can be expressed as the ratio, LD50/ED50.Compounds that exhibit large therapeutic indices are preferred. Whilecompounds that exhibit toxic side effects can be used, care should betaken to design a delivery system that targets such compounds to thesite of affected tissue to minimize potential damage to normal cellsand, thereby, reduce side effects.

The data obtained from cell culture assays and animal studies can beused to formulate a dosage range for use in humans. The dosage of suchcompounds lies preferably within a range of circulating concentrationsthat include the ED50 with little or no toxicity. The dosage can varywithin this range depending upon the dosage form employed and the routeof administration. For any compound used in the methods of theinvention, the therapeutically effective dose can be estimated initiallyfrom cell culture assays. A dose can be formulated in animal models toachieve a circulating plasma concentration range that includes the IC50(the concentration of the test compound that achieves a half-maximalinhibition of symptoms) as determined in cell culture. Such informationcan be used to more accurately determine useful doses in humans. Levelsin plasma can be measured, for example, by high performance liquidchromatography (HPLC). In general, the dose equivalent of a modulator isfrom about 1 ng/kg to 10 mg/kg for a typical subject.

The amount and frequency of administration of the compounds of theinvention and/or the pharmaceutically acceptable salts thereof will beregulated according to the judgment of the attending clinicianconsidering such factors as age, condition and size of the patient aswell as severity of the symptoms being treated. An ordinarily skilledphysician or veterinarian can readily determine and prescribe theeffective amount of the drug required to prevent, counter or arrest theprogress of the condition. In general it is contemplated that aneffective amount would be from 0.001 mg/kg to 10 mg/kg body weight, andin particular from 0.01 mg/kg to 1 mg/kg body weight. More specificallyit is contemplated that an effective amount would be to continuouslyinfuse by intravenous administration from 0.01 micrograms/kg bodyweight/min to 100 micrograms/kg body weight/min for a period of 12 hoursto 14 days. It may be appropriate to administer the required dose astwo, three, four or more sub-doses at appropriate intervals throughoutthe day. Said sub-doses may be formulated as unit dosage forms, forexample, containing 0.01 to 500 mg, and in particular 0.1 mg to 200 mgof active ingredient per unit dosage form.

Preferably, the pharmaceutical preparation is in a unit dosage form. Insuch form, the preparation is subdivided into suitably sized unit dosescontaining appropriate quantities of the active component, e.g., aneffective amount to achieve the desired purpose. The quantity of activecompound in a unit dose of preparation may be varied or adjusted fromabout 0.01 mg to about 1000 mg, preferably from about 0.01 mg to about750 mg, more preferably from about 0.01 mg to about 500 mg, and mostpreferably from about 0.01 mg to about 250 mg, according to theparticular application. The actual dosage employed may be varieddepending upon the requirements of the patient and the severity of thecondition being treated. Determination of the proper dosage regimen fora particular situation is within the skill of the art. For convenience,the total dosage may be divided and administered in portions during theday as required.

Medical Use

The compositions of this invention are useful for treating anycardiovascular disorder that will respond favorably to a decrease inblood pressure. These disorders include chronic hypertension,hypertensive crisis (an acute hypertensive emergency), acute congestiveheart failure, angina, acute myocardial infarction, left ventricularfailure, cerebrovascular insufficiency, and intracranial haemorrhage.Intravenous injection is a preferred method for treating acutecardiovascular disorders. Such a method would comprise administering atherapeutically effective amount of one or more compounds of the presentinvention to a subject in need thereof. Examples of acute cardiovasculardisorders include, but are not limited to, hypertensive crisis, toxemiaof pregnancy, and acute congestive heart failure.

Combination Therapies

The invention also provides a method of treating any cardiovascular orcardiorenal disorder by administering one or more of the compositions ofthe invention as described above in combination with other drugs for thetreatment of cardiovascular and/or cardiorenal disorders. These otherdrugs include diuretics such as furosemide; vasodilators such asnitroglycerin, nitroprusside, brain natriuretic peptide (BNP), oranalogues thereof; inotropes such as dobutamine; angiotensin convertinenzyme (ACE) inhibitors such as captopril and enalapril; blockers suchas carvedilol and propranolol; angiotensin receptor blockers (ARBs) suchas valsartan and candesartan; and/or aldosterone antagonists such asspironolactone.

In the combination therapies of the invention, one or more compositionsof the invention are coadministered with one or more drugs for thetreatment of cardiovascular and/or cardiorenal disorders to increaseefficacy of treatment of cardiovascular and/or cardiorenal disorders andto reduce side effects associated with high doses of these therapeutics.

The combination therapies of the invention described above havesynergistic and additive therapeutic effects. Synergy is defined as theinteraction of two or more agents so that their combined effect isgreater than the sum of their individual effects. For example, if theeffect of drug A alone in treating a disease is 25%, and the effect ofdrug B alone in treating a disease is 25%, but when the two drugs arecombined the effect in treating the disease is 75%, the effect of A andB is synergistic.

Additivity is defined as the interaction of two or more agents so thattheir combined effect is the same as the sum of their individualeffects. For example, if the effect of drug A alone in treating adisease is 25%, and the effect of drug B alone in treating a disease is25%, but when the two drugs are combined the effect in treating thedisease is 50%, the effect of A and B is additive.

An improvement in the drug therapeutic regimen can be described as theinteraction of two or more agents so that their combined effect reducesthe incidence of adverse event (AE) of either or both agents used inco-therapy. This reduction in the incidence of adverse effects can be aresult of, e.g., administration of lower dosages of either or both agentused in the co-therapy. For example, if the effect of Drug A alone is25% and has an adverse event incidence of 45% at labeled dose; and theeffect of Drug B alone is 25% and has an adverse event incidence of 30%at labeled dose, but when the two drugs are combined at lower thanlabeled doses of each, if the overall effect is 35% (an improvement, butnot synergistic or additive) and the adverse incidence rate is 20%,there is an improvement in the drug therapeutic regimen.

DEFINITIONS

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In the case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only not intended tobe limiting. Other features and advantages of the invention will beapparent from the following detailed description and claims.

For the purposes of promoting an understanding of the embodimentsdescribed herein, reference will be made to preferred embodiments andspecific language will be used to describe the same. The terminologyused herein is for the purpose of describing particular embodimentsonly, and is not intended to limit the scope of the present invention.As used throughout this disclosure, the singular forms “a,” “an,” and“the” include plural reference unless the context clearly dictatesotherwise. Thus, for example, a reference to “a composition” includes aplurality of such compositions, as well as a single composition, and areference to “a therapeutic agent” is a reference to one or moretherapeutic and/or pharmaceutical agents and equivalents thereof knownto those skilled in the art, and so forth. Thus, for example, areference to “a host cell” includes a plurality of such host cells, anda reference to “an antibody” is a reference to one or more antibodiesand equivalents thereof known to those skilled in the art, and so forth.

EXAMPLES

The following examples are illustrative, but not limiting, of themethods and compositions of the present invention. Other suitablemodifications and adaptations of the variety of conditions andparameters normally encountered in therapy and that are obvious to thoseskilled in the art are within the spirit and scope of the embodiments.

Example 1 Synthesis of Compounds

Peptides and intermediates described herein were prepared by thesolid-phase method of peptide synthesis. (cf. R. Merrifield J. Am. Chem.Soc. 1964, 85, 2149; M. Bodansky, “Principles of Peptide Synthesis.”Springer-Verlag, 1984.) The peptide synthesis and purificationprocedures employed were standard methods well described in the art,including, but not limited to, amino acid coupling procedures, washsteps, deprotection procedures, resin cleavage procedures, and ionexchange and HPLC purification methods using commercial automatedpeptide synthesizers and commercially available resins and protectedamino acids. More specifically, the peptides were synthesized from theirC-terminus by stepwise addition of Fmoc-protected amino acids(pre-activated or in situ activated) and deprotection of the Fmoc groupwith piperidine to an acid labile linker attached to an insolublesupport resin. Following synthesis, the resin bound peptide was sidechain-deprotected and detached from the resin with trifluoroacetic acidand cation scavengers. Peptides were purified by aqueous extraction orby precipitation from organic solvents such as ether or t-butyl methylether followed by centrifugation and decanting and/or by HPLC andlyophilization.

Example 2 B-Arrestin Recruitment Assay

The proximal event in β-arrestin function mediated by GPCRs isrecruitment to receptors following ligand binding and receptorphosphorylation by GRK's. Thus, the measure of β-arrestin recruitmentwas used to determine ligand efficacy for β-arrestin function.

B-arrestin-2 recruitment to the human and rat angiotensin 2 type 1receptor (human AT1R and rat AT1aR, respectively) was measured with thePathHunter™ β-arrestin assay (DiscoveRx Corporation, Fremont Calif.).Cells, plasmid(s), and detection reagent(s) were purchased fromDiscoveRx, and assays were performed per manufacturer's instructions.Human AT1R and rat AT1aR were cloned into the pCMV-ProLink vector,verified by sequencing, and transfected into PathHunter β-arrestinHEK293 cells. Stably transfected clonal cell lines were selected withHygromycin and G418. These clonal cell lines were used for allexperiments.

For assays, 25,000 cells were seeded per well in 96-well microplates involumes of 90 μL and allowed to grow overnight at 37° C. in 5% CO₂.Peptides of the invention were dissolved in deionized water to aconcentration of 1 mM. Peptides were then further diluted in assaybuffer (Hank's balanced salt solution with 20 mM HEPES) to add peptideto the cells to reach final concentrations ranging from 10 μM to 1 pM.Cells were then incubated for 60 minutes at 37° C. in 5% CO₂, followedby addition of 50 μL of PathHunter Detection Reagent to each well. Themicroplates were then incubated at room temperature for 60 minutes, andthen luminescence was measured using a NOVOstar microplate readerpurchased from BMG Labtech. B-arrestin-2 recruitment to receptors wasmeasured as relative luminescence intensity expressed in arbitraryunits. Results are displayed in Table 2 below.

Example 3 IP1 Accumulation Assay

A secondary measure of G protein coupling efficacy was also performed.IP3 is generated by activation of phospholipase C by Gα-q. IP3 isdegraded to IP1, which can be forced to accumulate in cells by blockingdegradation with lithium chloride. Thus we measured accumulation of IP 1to determine ligand efficacy for G protein activation.

IP1 accumulation generated by human and rat angiotensin 2 type 1receptor (human AT1R and rat AT1aR, respectively) was measured withIP-One Tb kits purchased from Cisbio and used per the manufacturer'sinstructions. Clonal stably transfected cell lines expressing humanAT1TR or rat AT1aR were used for all experiments.

For assays, 4,000 cells were seeded per well in 384-well small-volumemicroplates in volumes of 20 uL and allowed to grow overnight at 37° C.in 5% CO₂. Cell growth media was then replaced with stimulation buffersupplied by Cisbio containing 50 mM lithium chloride. PeptidesTRV-120,001 through TRV-120,035 were dissolved in deionized water to aconcentration of 1 mM. For agonist detection, peptides were then furtherdiluted in stimulation buffer to add peptide to the cells to reach finalconcentrations ranging from 10 uM to 1 pM. For antagonist detection,peptides were then further diluted in stimulation buffer to add peptideto the cells to reach final concentrations ranging from 10 uM to 1 pM,and followed by addition of 10 nM AngII. Following addition of peptides,cells were incubated at 37° C. in 5% CO₂ for 30 minutes and then lysedwith detection reagents added per manufacturer's instructions.Microplates were incubated for 60-90 minutes and then time-resolvedfluorescence intensities were measured using a PHERAstar Plus microplatereader from BMG Labtech. IP1 accumulation was measured as change inratio of time-resolved fluorescent intensities measured at 665 nm and620 nm. Results are displayed in Table 2 below.

TABLE 2 Biological activity. human AT1R rat AT1aR b-arrestin2 IP Oneb-arrestin2 IP One Max Max Max Max # EC50 (M) (%) EC50 (M) (%) EC50 (M)(%) EC50 (M) (%) AngII 9.7E−09 99.0 2.6E−09 96.9 6.8E−09 97.0 1.3E−0998.1 Valsartan inactive inactive inactive  2 4.5E−06 24.9 inactiveinactive 3.6E−07 33.8  3 4.5E−07 36.5 inactive 1.2E−07 67.1 4.1E−08 19.6 5 1.6E−06 39.4 6.5E−08 18.1 1.9E−06 61.7 9.6E−08 24.8  6 9.2E−07 12.11.1E−07 14.1 inactive 5.9E−08 21.9  7 1.1E−08 63.0 inactive 7.5E−09 86.56.0E−09 9.3  8 2.1E−07 36.9 inactive 2.0E−07 41.8 3.5E−08 11.3  99.2E−09 69.2 inactive 4.8E−09 74.0 6.6E−09 9.5 10 1.6E−07 55.2 inactive1.8E−07 60.3 2.2E−07 13.3 11 3.4E−09 58.9 inactive 6.1E−09 80.7 3.5E−0924.4 12 3.9E−07 19.9 1.9E−07 20.7 1.2E−07 34.4 7.9E−08 44.9 13 4.6E−0643.4 inactive 4.3E−05 165.3 Inactive 14 8.0E−09 59.3 1.6E−07 13.51.4E−08 79.0 3.2E−08 19.3 15 8.2E−07 44.3 2.5E−07 19.3 2.3E−07 62.17.7E−08 51.1 16 8.8E−07 56.9 inactive 1.4E−07 76.9 9.2E−08 24.4 193.1E−08 67.9 inactive 1.8E−08 91.2 Inactive 20 inactive inactive 3.6E−0686.6 Inactive 21 3.3E−08 64.4 inactive 1.3E−08 82.7 Inactive 22 2.7E−0754.5 inactive 2.7E−08 74.3 2.5E−07 5.2 23 4.4E−08 61.2 inactive 3.9E−0884.9 1.1E−08 10.3 24 4.8E−07 61.3 inactive 6.3E−08 74.1 7.4E−09 15.1 252.1E−08 57.9 inactive 1.5E−08 77.0 7.3E−09 14.1 26 7.5E−08 51.9 inactive3.8E−08 77.3 Inactive 27 1.7E−08 58.1 inactive 1.6E−08 84.2 Inactive 282.0E−08 82.9 inactive 1.9E−08 91.4 6.4E−08 24.6 29 5.9E−07 43.3 inactive2.0E−07 64.8 Inactive 30 9.2E−09 68.2 inactive 8.2E−09 80.4 Inactive 311.0E−07 58.1 inactive 3.7E−08 75.9 1.3E−08 6.2 32 7.9E−08 55.6 inactive4.3E−08 80.2 2.4E−08 6.8 33 4.0E−08 54.9 inactive 3.1E−08 86.9 Inactive34 2.5E−08 53.0 inactive 3.2E−08 81.7 Inactive 35 1.2E−08 67.9 2.7E−097.1 9.3E−09 79.8 1.2E−08 30.5 36 1.2E−06 39.5 inactive 1.9E−06 32.81.0E−07 19.2 37 1.7E−06 62.3 inactive 1.9E−06 60.7 2.4E−07 14.7 386.8E−07 43.1 inactive 2.5E−06 66.4 9.2E−07 20.8 44 8.0E−08 47.3 inactive6.5E−08 71.9 8.2E−09 14.6 57 4.3E−07 42.06 3.6E−09 10.03 4.5E−07 64.383.3E−07 35.26 58 3.3E−07 48.67 inactive 2.6E−07 81.1 1.4E−06 9.937 591.4E−06 19.49 inactive 8.2E−07 27.84 Inactive 60 3.4E−07 44.02 inactive1.8E−07 79.27 Inactive 61 1.2E−06 37.55 inactive 2.1E−06 60.39 Inactive62 1.3E−06 24.68 inactive 3.9E−06 44.19 Inactive 63 5.3E−07 35.93inactive 5.3E−07 50.13 Inactive 64 1.2E−06 20.39 inactive 8.7E−07 26.95Inactive 65 2.0E−06 37.8 inactive 2.8E−06 67.27 Inactive 66 6.7E−0630.56 inactive 1.0E−05 58.54 Inactive 67 1.0E−07 100.6 inactive 6.3E−08117 Inactive 68 6.3E−07 39.2 inactive 4.0E−07 59.2 2.5E−05 21.2 691.3E−08 53.9 inactive 1.0E−08 88.3 Inactive 70 1.6E−07 29.1 inactive7.9E−08 55.6 Inactive 71 4.0E−07 52.7 inactive 1.6E−07 81.7 Inactive 726.3E−08 62.6 inactive 2.5E−08 88.6 Inactive 73 5.0E−06 46.8 inactive4.0E−06 77.4 2.5E−06 20.8 74 2.5E−07 47.8 inactive 1.0E−07 59.0 Inactive75 2.5E−08 55.0 inactive 1.3E−08 79.3 2.5E−05 26.7 76 1.6E−06 43.0inactive 6.3E−07 55.5 Inactive 77 5.0E−08 97.5 inactive 4.0E−08 110.5Inactive 78 1.0E−08 71.5 inactive 5.0E−09 91.3 Inactive 79 7.9E−09 64.8inactive 7.9E−09 93.6 Inactive 80 1.0E−06 58.5 inactive 5.0E−07 82.9Inactive 81 1.0E−05 32.6 inactive 1.0E−05 54.3 Inactive 82 3.2E−07 38.3inactive 1.3E−07 60.6 Inactive 83 2.0E−07 34.3 inactive 5.0E−08 70.2Inactive 84 3.2E−08 50.5 inactive 1.6E−08 80.6 Inactive 85 1.6E−06 51.15.0E−07 75.5 1.6E−05 23.4 Note: inactive means that EC50 was greaterthan 10 uM.

Example 4 Calcium Mobilization Assay

G protein efficacy can be measured in many ways. GPCRs that couple tothe G_(q) subclass of heterotrimeric g proteins activate a wide array ofsignal transduction when activated by agonists. One of the most commonlymeasured pathways is activation of phospholipase C by Galpha-q, whichcleaves phosphatidylinositol bisphosphate to release IP₃; IP₃ in turnreleases calcium to the cytosol from intracellular stores via the IP₃receptor. Thus we measured intracellular free calcium to determineligand efficacy for G protein activation.

Intracellular free calcium generated by human and rat angiotensin 2 type1 receptor (human AT1R and rat AT1aR, respectively) was measured withFluo-4 NW kits purchased from Invitrogen and used per the manufacturer'sinstructions. Clonal stably transfected cell lines expressing humanAT1TR or rat AT1aR were used for all experiments.

For assays, 25,000 cells were seeded per well in 96-well microplates involumes of 90 uL and allowed to grow overnight at 37° C. in 5% CO₂.Fluo-4 NW dye was mixed with probenecid and assay buffer (Hank'sbalanced salt solution with 20 mM HEPES), and cell growth media wasreplace with this mixture, followed by incubation for 30-45 minutes at37° C. in 5% CO₂. Peptides TRV-120,001 through TRV-120,035 weredissolved in deionized water to a concentration of 1 mM. Peptides werethen further diluted in assay buffer (Hank's balanced salt solution with20 mM HEPES) to add peptide to the cells to reach final concentrationsranging from 10 uM to 1 pM. Peptide was added to cells whilefluorescence intensity was measured using a NOVOstar microplate readerpurchased from BMG Labtech. Calcium mobilization was measured asrelative fluorescence intensity expressed as fold over basal at 5seconds and 20 seconds after ligand addition.

Example 5 Evaluation of SEQ ID NO:27 in Normal Rats

The effects of SEQ ID NO:27 on vascular and cardiac function were testedby i.v. infusion at doses ranging from 0.1-10 μg/kg/min in preliminarydosing experiments in normal anesthetized rats. Various hemodynamicmeasurements were made including mean arterial pressure, heart rate andpressure volume relationships. SEQ ID NO:27 produced a dose-dependentdecrease in mean arterial pressure with little to no effect on HR. Inaddition, SEQ ID NO:27 increased the slope of the end systolic pressurevolume relationship and preserved pre-recruitable stroke work, resultingin preservation of stroke volume in the background of a drop invasoconstriction.

Example 6 Evaluation of SEQ ID NO:27 in a Paced Dog Model of Acute HeartFailure

SEQ ID NO:27 (0.01, 0.1, 1, 10 and 100 mcg/kg/min dose escalation, 30minutes each dose) was also dosed in the paced heart failure model. Inthe paced dog model, pacemakers are implanted and the dog hearts arepaced for ten days at a rate of 240 beats per minute, resulting inreduced left ventricular systolic function, right-side congestion, andan elevation in the renin-angiotensin system activity. In the heartfailure dogs SEQ ID NO:27 produced a dose-dependent decrease in meanarterial pressure, systemic vascular resistance, pulmonary capillarywedge pressure, and right arterial pressure, and cardiac output waspreserved in these animals. At the level of the kidney, there was adose-dependent increase in renal blood flow resulting in a significantdrop in renal vascular resistance. Urine sodium excretion modestlyincreased with urine output, urine potassium, and glomerular filtrationrate being maintained.

While the invention has been described with reference to particularlypreferred embodiments and examples, those skilled in the art recognizethat various modifications may be made to the invention withoutdeparting from the spirit and scope thereof.

All of the above U.S. patents, U.S. patent application publications,U.S. patent applications, foreign patents, foreign patent applicationsand non-patent publications referred to in this specification and/orlisted in the Application Data Sheet are incorporated herein byreference, in their entirety.

What is claimed is:
 1. A peptide or peptide mimetic comprising thesequence of: Sar-Arg-Val-Ww-Zz-His-Pro-Cc, wherein Ww is L-tyrosine,3-hydroxy-L-tyrosine, 3-fluoro-L-tyrosine, 2,6-difluoro-L-tyrosine,3-nitro-L-tyrosine, 3,5-dinitro-L-tyrosine, 3-chloro-L-tyrosine,2,6-dimethyl-L-tyrosine, 4-fluorophenyl-L-alanine,3,5-dibromo-L-tyrosine, or O-allyl-L-tyrosine, Zz is L-valine orL-isoleucine, and Cc is D-alanine.
 2. The peptide of claim 1, whereinthe peptide or peptide mimetic comprises the sequence of SEQ ID NO:69.3. The peptide of claim 1, wherein the peptide or peptide mimeticcomprises the sequence of SEQ ID NO:67.
 4. The peptide or peptidemimetic of claim 1, wherein Ww is selected from the group consisting ofL-tyrosine, 3-hydroxy-L-tyrosine, 3-fluoro-L-tyrosine,2,6-difluoro-L-tyrosine, 3-nitro-L-tyrosine, 3,5-dinitro-L-tyrosine, and3-chloro-L-tyrosine.
 5. The peptide or peptide mimetic of claim 1,wherein the peptide or peptide mimetic comprises the sequence of SEQ IDNO:27, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ IDNO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, or SEQ IDNO:76.
 6. The peptide of claim 1, wherein the peptide comprises thesequence of SEQ ID NO:
 69. 7. The peptide of claim 1, wherein thepeptide comprises the sequence of SEQ ID NO:67, SEQ ID NO:68, SEQ IDNO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ IDNO:74, SEQ ID NO:75, or SEQ ID NO:76.
 8. The peptide of claim 1, whereinZz is L-isoleucine.
 9. The peptide of claim 4, wherein Zz isL-isoleucine.
 10. A pharmaceutical composition comprising one or morepeptides or peptide mimetics of claim 1 and a pharmaceuticallyacceptable carrier.
 11. The pharmaceutical composition of claim 10,wherein the pharmaceutically acceptable carrier is pure sterile water,phosphate buffered saline or an aqueous glucose solution.
 12. Thepharmaceutical composition of claim 10, wherein the pharmaceuticalcomposition comprises the peptide comprising a sequence of SEQ ID NO:27,SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71,SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, or SEQ ID NO:76.13. The pharmaceutical composition of claim 10, wherein thepharmaceutical composition comprises the peptide comprising a sequenceof SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71,SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, or SEQ ID NO:76.14. The pharmaceutical composition of claim 10, wherein thepharmaceutical composition comprises the peptide comprising a sequenceof SEQ ID NO:69.
 15. The pharmaceutical composition of claim 10, whereinthe pharmaceutical composition comprises sterile water.
 16. The peptideor peptide mimetic of claim 1, wherein Ww is selected from the groupconsisting of 3-hydroxy-L-tyrosine, 3-fluoro-L-tyrosine,2,6-difluoro-L-tyrosine, 3-nitro-L-tyrosine, 3,5-dinitro-L-tyrosine, and3-chloro-L-tyrosine.